Methods for identification of modulators of OSGPR114 or OSGPR78 activity, and their use in the treatment of disease

ABSTRACT

This invention relates to the identification of LPA as a ligand for the G-protein coupled receptors OSGPR114 and OSGPR78. The invention is directed to new methods for screening candidate drugs for their ability to modulate the activity of OSGPR114 or OSGPR78, and new pharmaceutical agents identified by these methods. It is also directed to the use of such agents in the manufacture of medicaments for the treatment of OSGPR114 or OSGPR78 mediated diseases, and methods of treating diseases such as cancers by administering to an individual a therapeutic amount of a modulator of OSGPR114 or OSGPR78 identified by these methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/482,964, filed Jun. 27, 2003, which is herein incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO SEQUENCE LISTING

A complete sequence listing section is included herein.

BACKGROUND OF THE INVENTION

This invention relates to the identification of lysophosphatidic acid(LPA) as a ligand for the G-protein coupled receptors OSGPR114 andOSGPR78, and is directed to in vitro methods for screening candidatedrugs for their ability to modulate the activity of OSGPR114 or OSGPR78,and to methods of treating disease by administering to an individual atherapeutic amount of a modulator of OSGPR114 or OSGPR78.

G-protein coupled receptors (GPCRs) are a super-family of membranereceptors that mediate a wide variety of biological functions. Uponbinding of extracellular ligands, GPCRs interact with a specific subsetof heterotrimeric G proteins that can, in their activated forms, inhibitor activate various effector enzymes and/or ion channels. All GPCRs arepredicted to share a common molecular architecture consisting of seventransmembrane helices linked by alternating intracellular andextracellular loops. The extracellular receptor surface has been shownto be involved in ligand binding whereas the intracellular portions areinvolved in G-protein recognition and activation. Different G-proteinalpha-subunits, and beta-gamma subunit complexes, preferentiallystimulate or inhibit particular effector molecules to modulate variousbiological functions in a cell. Typical effector molecules includeadenylate cyclase, phospholipases C and A2, cGMP phosphodiesterase-γ,and potassium, sodium and calcium channels. Additional regulation ofGPCR activity is thought to occur via receptor oligomerization andinteraction with the protein β-arrestin (e.g. see Rocheville, M. et. al.(2000) Science 288:154-157; Gether, U. (2000) Endocrine Reviews21:90-113; Luttrel, L. M. et. al. (1999) Science 283:655-661). G-proteincoupled receptors are found ubiquitously in all cell types withinmammalian organisms. Many therapeutic agents targeting GPCR receptorshave been successfully introduced onto the market, thereby establishingtheir value as targets for drug discovery and development (e.g. Wise, A.et al. 2002, DDT, 7:235-246). Over 30% of clinically marketed drugs areactive on GPCRs.

It has been estimated that for about 40% of GPCRs in the human genome(excluding sensory receptors) the ligand remains unknown. Such GPCRs arecommonly referred to as “orphan” receptors. For example, the primarystructures of a human isoform of the orphan GPCRs OSGPR114 and OSGPR78,or closely related GPCRs, have been described in recent patentapplications (e.g. OSGPR114 in WO 01/90187, WO 01/87937, WO 01/42288, WO01/77326, WO 01/48189, WO 01/31014, WO 01/04292, WO 01/02563, WO00/23588, WO 00/31258, WO 00/50458 and EP 1090926 A1; and OSGPR78 in WO96/30406). The DNA and amino acid sequences for OSGPR114 and OSGPR78have also been described in the scientific literature. A chicken homologof OSGPR78 was originally identified in chicken activated T-cells andnamed 6H1 by Kaplan (Kaplan et. al. (1993) J. Immunol. 151(2):628-636).Webb (Webb et. al. (1996) Biochem. Biophys. Res. Commun. 219(1):105-110)followed the disclosure of the sequence with a proposal that thereceptor bound to ATP, and therefore named it P2Y5 as the fifth memberof the purinergic GPCR P2Y family. However, subsequent studies (Li et.al. (1997) Biochem. Biophys. Res. Commun. 236(2):455-60); andexperiments described herein) could not find evidence that the receptorwas in fact activated by nucleotides, thus calling into question theclassification as a P2Y receptor. The human OSGPR78 was sequencedearlier, while sequencing the complete genomic sequence of the humanretinoblastoma susceptibility gene (Toguchida et al. (1993) Genomics 17:535-543), but it was not appreciated until later that the human P2Y5receptor is encoded in the intron 17 of the retinoblasoma gene (Herzoget al. 1996, Genome research 6: 858-61; Bohm et al. 1997, Genbank entrylocus AAB62190, direct submission to Genbank). In the scientificliterature, OSGPR114 was originally identified by White (White et. al.(2000) Nat. Genet. 26, 345-348) and named gpr92. Lee (Lee et. al. (2001)Gene 275(1):83-91) also disclosed the sequence.

No information outwith nucleic acid and amino acid sequence homologiesand expression patterns for OSGPR114 or OSGPR78 have been described inthe scientific literature, e.g. no activating ligand has beenidentified. Similarly, in the above patent applications OSGPR114 andOSGPR78 are described as tools for identifying drugs for the treatmentof a variety of pathophysiological conditions. However, none of theseapplications identify a function for either of these GPCRs, or describesa ligand that binds to either receptor and modulates its activity.

It has been previously shown that LPA has the ability to modulate cellmotility and growth and to stimulate tumor growth; to modulate thedevelopment and regulation of the cardiovascular system including acontribution to artherosclerosis and a role in wound healing and tissueregeneration; to regulate the differentiation of multiple cell typesincluding the induction of differentiation of preadipocytes intoadipocytes; and to have influence over the physiology andpathophysiology of the reproductive tracts of males and of females.Prior to the work described herein, the effects of LPA in these andother systems were thought to be exclusively the result of interactionwith the LPA receptors LPA-1, -2 and -3, previously known as Edg-2, -4and -7 (Chun, J., et al. (2002) Pharmacol. Reviews 54:265-269).

LPA is one of the simplest phospholipids found in nature and consists ofa glycerol moiety with a fatty acid backbone at the sn1 (or sn2)position, a phosphate group at the sn3 position and a hydroxyl at thesn2 (or sn1) site. It has been shown that endogenous LPA species cancontain multiple fatty acids. These fatty acids may vary in their chainlength, the amount of unsaturation/saturation and may consist of an acylor alkyl linkage. It has been shown that in some cases, the predominantspecies of LPA may vary between tissues and/or cell types and isinfluenced by the available precursor lipids within a particular cell ortissue. The biosynthetic pathways and metabolic pathways of LPA may alsovary between cells and are only moderately well characterized.Intracellular and extracelullar synthetic and degradative pathways forLPA are also different, as are the physiological roles of LPA on eitherside of the cell membrane.

Amongst the multiple known biological roles of LPA, much of thescientific literature attention has been focused on the ability of LPAto act as a proliferative signal to cells of multiple origins,especially malignant cells. In addition to acting as a growth stimulatorto cancer cells, LPA has been demonstrated by multiple studies to act asa motility factor and an angiogenic factor in carcinogenesis and cancerprogression. It has been shown to increase the invasive capacity ofcancer cells, and to have an important role in increasing the metastaticpotential of tumors. It is therefore known that LPA is stronglyimplicated in controlling and contributing towards virtually all aspectsof malignant disease. One of the original studies on the role of LPA incancer identified “ovarian cancer activating factor” as LPA (Xu et. al.1995 Clin. Cancer Res. 1(10): 1223-32). Additionally, increased tumorproduction of LPA has been observed and the enzyme responsible for thisshown to be upregulated in multiple cancers. Further validation of therole of LPA in cancer disease is shown by the fact that induction orexpression of enzymes that degrade LPA do not only prevent the activityof LPA in disease progression in vitro, but also dramatically reducetumor growth in vivo (Tanyi et. al. 2003 Cancer Res. 63(5):1073-1082).Additionally, LPA levels in the blood, and in ascites, have been shownto be significantly higher in patients with ovarian cancer than inpatients who do not have ovarian cancer. The degree of this elevation inblood LPA has been correlated with tumor malignancy. As well as inducingthe growth of ovarian cancer cells, LPA also increases their motilityand invasiveness and at concentrations present in ascites, preventscisplatin-induced apoptosis. In summary, there is an extensive body ofpublic literature that conclusively demonstrates that LPA signaling isaberrant in multiple cancers. Cancer types that have been implicated asinvolving dysregulated LPA signaling, in addition to ovarian cancer,include cancers of the lung, prostate, pancreas, colon, breast,esophagus, kidney and stomach, and glioma, lymphoma, leukemia andmelanoma.

The molecular mechanisms behind the involvement of LPA in cancer are thesubject of multiple reviews (e.g. Fang et. al. (2000) Ann. N.Y. Acad.Sci. 905:188-208; Fujita et. al. (2003) Cancer Letts 192:161-9; Ericksonet. al. (2001) Prostaglandins and other Lipid Mediators 64: 63-81; Daaka(2002) Biochim. Biophys. Acta 1582: 265-269; Fukushima et. al. (2001)Ann. Rev. Pharm. Toxicol. 41:507-34). Although LPA is known to act asboth an intracellular and extracellular signaling moiety, most studiesinvestigating the role of LPA in cancer have focused on its role as anautocrine and paracrine growth factor, predominantly stimulating thegrowth of cancer cells and tumors. Such extracellular signaling pathwayshave also been shown to be intimately involved in increases in cancercell motility, invasiveness, angiogenesis and metastasis resulting fromLPA administration to cancer cells. The extracellular signaling of LPAis known to be transduced to the cell interior via LPA specific GPCRs.Such receptors have been described in the literature, and there arethree that have been well characterized to date, LPA1, LPA2 and LPA3.They were previously named Edg2, Edg4 and Edg7 due to their highhomology with other phospholipid receptors Edg 1, 3, 5, 6 and 8, (whichare now known as Sphingosine 1-phosphate (SIP) receptors 1 to 5). Edg isan acronym for “endothelial differentiation gene”. Very recently afourth LPA receptor, p2y9/GPR23, has also been reported (Noguchi et. al.(2003) J. Biol. Chem. April 30, electronic manuscript M302648200 aheadof print). p2y9/GPR23 is only distantly related to LPA1, LPA2 and LPA3.Additionally, activation of GPCRs by LPA is also known to transactivateother growth factors involved in cancer development, such as theepidermal growth factor receptor and the platelet-derived growth factorreceptor (for review see Wu and Cunnick (2002) Biochim. Biophys. Acta.1582(1-3): 100-106). The effects of LPA on mitogenesis and survivallikely involve activation of ERK and transactivation of other growthfactor receptors, whereas the effects on motility and invasion probablyoccur through Rho-based signaling, probably via coupling of the receptorto Galpha 12/13. The effects on angiogenesis probably occur through theinduction of proangiogenic factors such as VEGF.

Despite the considerable body of literature in this area, a completeunderstanding of LPA action in modulating cell proliferation and tumorgrowth at the molecular level has not yet been achieved. Consequently,without a full understanding of its mechanism of action, there areconsiderable problems associated with developing compounds thatantagonize such effects of LPA. To help alleviate this problem, thesurprising discovery described herein that certain LPA compounds areligands of OSGPR114 and OSGPR78, and therefore that the latter are novelmembers of the family of LPA-activated GPCRs, suggests that the effectof LPA as a physiological modulator of various physiological systems isnot limited to interaction with the previously known LPA receptors.Thus, this discovery provides an additional mechanism of action for LPA,additional targets for therapeutic modulation, and thus a basis forfurther assay and drug development. Several compounds are already underdevelopment as modulators of the activity of other LPA receptors (e.g.EP 1258484 A1, WO 02/29001, US 2003/0027800 A1 and U.S. Pat. No.6,380,177; Fischer, D. J., et al. (2001) Mol Pharmacol. 60(4):776-84;Hasegawa, Y., et al. (2003) J. Biol. Chem. 278(14):11962-9; Heise, C.E., et al. (2001) Mol. Pharmacol. 60(6):1173-80; Hooks, S. B., et al.(2001) J Biol. Chem. 276(7):4611-21; Hopper, D. W., et al. (1999) J.Med. Chem. 42(6):963-70; Tigyi, G. (2001) Mol Pharmacol. 60(6): 1161-4;Yokoyama, K., et al. (2002). Biochim. Biophys. Acta 1582(1-3): 295-308;Gueguen, G., et al. (1999) Biochemistry 38(26): 8440-50; Lynch, K. R.and T. L. Macdonald (2002). Biochim. Biophys. Acta 1582(1-3): 289-94;Sardar, V. M., et al. (2002) Biochim. Biophys. Acta 1582: 309-307 andVirag, T., et al. (2003) Mol Pharmacol. 63(5):1032-42). Some of thesecompounds have been found to have selective activity on one or more LPAreceptors, while others have equivalent activity on all LPA receptorstested.

SUMMARY OF THE INVENTION

The present invention is based on the finding that both of the G-proteincoupled receptors OSGPR114 and OSGPR78 are able to act as receptors forlysophosphatidic acid (LPA) and that cells transfected to expressOSGPR114 or OSGPR78 gain the ability to elicit Gi/o (i.e. Gi and/or Go)or other G-protein mediated responses following exposure to LPA.Suitable LPA compounds include, but are not limited to, myristoyllysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid, or afunctional analog or homolog of one of these compounds. Identificationof a ligand for OSGPR114 or OSGPR78 therefore facilitates thedevelopment of screening methods for identifying modulators of theOSGPR114 or OSGPR78 receptor.

Accordingly the invention further provides a method for identifyingagents which modulate the activity of OSGPR114 or OSGPR78 receptor,which comprises determining whether the test agent interacts withOSGPR114 or OSGPR78. The method may comprise the use of OSGPR114 orOSGPR78 in combination with an LPA ligand. The invention furthercomprises the use of agents identified using the method of the inventionin the treatment of diseases mediated by OSGPR114 or OSGPR78 and theiruse in the manufacture of a medicament for the treatment of OSGPR114 orOSGPR78 mediated diseases. Accordingly, the invention further provides amethod of treatment of diseases or conditions mediated by OSGPR114 orOSGPR78 in an individual, which comprises the administration of atherapeutically effective amount of an OSGPR114 or OSGPR78 receptormodulator. The invention also provides the use of a modulator ofOSGPR114 or OSGPR78 in the manufacture of a medicament for the treatmentof diseases or conditions mediated by OSGPR114 or OSGPR78.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Nucleotide sequence encoding human OSGPR114 (FIG. 1A, SEQ I.D.NO:1) and OSGPR78 (FIG. 1B, SEQ I.D. NO:2) receptors.

FIG. 2: Deduced amino acid sequence of human OSGPR114 (SEQ I.D. NO:3)and OSGPR78 (SEQ I.D. NO:4) receptors

FIG. 3: OSGPR78 and OSGPR114 expression profile in human cell lines.Data is expressed as a ratio of TFIIB expression levels in the same celllines and the experiment conducted as detailed in the Materials andMethods section.

FIG. 4: OSGPR114 gene expression in matched pairs of human tumors. Datais expressed as a ratio of TFIIB expression levels in the same tissuesand the experiment conducted as detailed in the Materials and Methodssection.

FIG. 5: OSGPR78 gene expression in matched pairs of human tumors. Datais expressed as a ratio of TFIIB expression levels in the same tissuesand the experiment conducted as detailed in the Materials and Methodssection.

FIG. 6: Effects of LPA at OSGPR78. The experiment was conducted asdetailed in the Materials and Methods section. Briefly, engineered yeastcells expressing different Galpha chimeric subunits and two reportergenes, URA-Fus1p-LacZ and TRP-Fus1p-LacZ were incubated with LPA. Theyeast cells were tested with and without OSGPR78 expression.β-galactosidase activity was measured and the result expressed as afold-induction over basal β-galactosidase activity. LPA stimulation offluorescence was only observed in receptor transformed yeast cells andnot in vector transformed yeast cells or in yeast cells of similarchimeric backgrounds expressing other receptors.

FIG. 7: Effects of different LPA species at OSGPR114. The experiment wasconducted as detailed in the Materials and Methods section. Briefly,engineered yeast cells expressing a Galpha chimeric subunit and tworeporter genes, URA-Fus1p-LacZ and TRP-Fus1p-LacZ were incubated withLPA. The yeast cells were tested with and without OSGPR114 expression.β-galactosidase activity was measured and the result expressed as afold-induction over basal β-galactosidase activity. LPA stimulation offluorescence was only observed in receptor transformed yeast cells andnot in vector transformed yeast cells or in yeast cells of similarchimeric backgrounds expressing other receptors.

FIG. 8: Specific binding of [3H]LPA (14 nM) in cell membranes derivedfrom OSGPR114-stably tranfected CHO-K1 cells. The experiment wasconducted as described in the Materials and Methods section and the dataare shown are the result of subtracting CHO-K1 parental cell membranespecific binding from CHO-OSGPR114 specific binding.

FIG. 9: Stimulation of GTPγS binding in cell membranes prepared fromOSGPR114-CHO and CHO-K1 cells. The experiment was conducted as describedin the Materials and Methods section.

FIG. 10: Effects of LPA on intracellular calcium levels in CHO-K1 andCHO-OSGPR114 cells. The experiments were conducted in the presence andabsence of pertussis toxin and as described in the Materials and Methodssection.

FIG. 11: Oligonucleotides designed for PCR cloning of human OSGPR114 andOSGPR78 (SEQ I.D. NOS:5-8). Oligonucleotides for the human homolog werebased on the predicted sequence based on human genomic sequencinginformation.

FIG. 12: Oligonucleotides designed for quantitative RT-PCR usingfluorogenic probe for OSGPR114, OSGPR78 and TFIIB (SEQ I.D. NOS:9-17).

FIG. 13: OSGPR114 and OSGPR78 expression profile in normal humantissues. Data is expressed as a ratio of TFIIB expression levels in thesame tissues and the experiment conducted as detailed in the Materialsand Methods section.

FIG. 14: Effect of LPA on proliferation and survival in HCT-8 cells inthe presence and absence of serum. Data are means+/−s.e. calculated fromtwo experiments conducted in triplicate. The y-axis units are astandardized expression of fluorescence or luminescence counts.

FIG. 15: Effect of transfection of non-specific, OSGPR114 and PLK1siRNAs on HCT-8 cell proliferation and apoptosis as measured using BrDUand Apo-One caspase assays respectively. All data are normalized tocells not treated with siRNA, which was defined as equal to 1, andstatistical comparisons (One-way ANOVA, Dunnett's post-hoc) were madebetween transfection of target genes and transfection of non-specificsiRNA controls. Compared to non-specific siRNA, both OSGPR114 siRNA andthe positive control PLK1 siRNA inhibited proliferation and inducedapoptosis in HCT-8 cells 72 h after transfection.

FIG. 16: Effect of transfection of non-specific, OSGPR114, OSGPR78 andPLK1 siRNAs on KLE cell proliferation and apoptosis as measured usingBrDU and Apo-One caspase assays respectively. All data are normalized tocells not treated with siRNA, which was defined as equal to 1, andstatistical comparisons (One-way ANOVA, Dunnett's post-hoc) were madebetween transfection of target genes and transfection of non-specificsiRNA controls. Compared to non-specific siRNA, OSGPR114 siRNA and PLK1significantly inhibited proliferation in KLE cells 72 h aftertransfection. OSGPR78 siRNA also inhibited proliferation in these cellsalthough the difference was not significant statistically. AdditionallyPLK1 siRNA induced apoptosis of KLE cells whereas OSGPR114 and OSGPR78had no statistical effect.

FIG. 17: Effect of transfection of non-specific, OSGPR78 and PLK1 siRNAson A2058 cell proliferation and apoptosis as measured using BrDU andApo-One caspase assays respectively. All data are normalized to cellsnot treated with siRNA, which was defined as equal to 1, and statisticalcomparisons (One-way ANOVA, Dunnett's post-hoc) were made betweentransfection of target genes and transfection of non-specific siRNAcontrols. Compared to non-specific siRNA, OSGPR78 and PLK1 siRNAssignificantly inhibited proliferation in A2058 cells 48 h aftertransfection. Both siRNAs induced apoptosis though only PLK1 wassignificantly significant.

FIG. 18: Effect of transfection of non-specific, OSGPR114, OSGPR78 andPLK1 siRNAs on HCT-116 cell proliferation and apoptosis as measuredusing Cell-titer Glo and Apo-One caspase assays respectively. All dataare normalized to cells not treated with siRNA, which was defined asequal to 1, and statistical comparisons (One-way ANOVA, Dunnett'spost-hoc) were made between transfection of target genes andtransfection of non-specific siRNA controls. Compared to non-specificsiRNA, OSGPR114 and PLK1 siRNAs significantly inhibited proliferationand induced apoptosis in HCT-116 cells 72 h after transfection.

FIG. 19: Effect of transfection of non-specific, OSGPR114, OSGPR78 andPLK1 siRNAs on H460 cell proliferation and apoptosis as measured usingCell-titer Glo and Apo-One caspase assays respectively. All data arenormalized to cells not treated with siRNA, which was defined as equalto 1, and statistical comparisons (One-way ANOVA, Dunnett's post-hoc)were made between transfection of target genes and transfection ofnon-specific siRNA controls. Compared to non-specific siRNA, OSGPR114,OSGPR78 and PLK1 siRNAs significantly inhibited proliferation andinduced apoptosis in H460 cells 72 h after transfection.

FIG. 20: Effect of transfection of non-specific, OSGPR114, OSGPR78 andPLK1 siRNAs on MDAH-2774 cell proliferation and apoptosis as measuredusing BrDU and Apo-One caspase assays respectively. All data arenormalized to cells not treated with siRNA, which was defined as equalto 1, and statistical comparisons (One-way ANOVA, Dunnett's post-hoc)were made between transfection of target genes and transfection ofnon-specific siRNA controls. Compared to non-specific siRNA, OSGPR114,OSGPR78 and PLK1 siRNA inhibited proliferation in MDAH-2774 cells 72 hafter transfection. The effect of OSGPR78 siRNA was not significantstatistically.

FIG. 21: Measurement of gene knockdown of OSGPR114 by OSGPR114 siRNA asmeasured by quantitative RT-PCR. OSGPR114 mRNA was reduced approximately86% whereas Edg4, another LPA receptor expressed by HCT-8 cells was notsignificantly affected. Measurement of protein knockdown was notpossible since an antibody for OSGPR114 does not presently exist.However, using an identical transfection protocol in HCT-8 cells, LaminB2 siRNA reduced Lamin B2 protein by approximately 60% as measured byWestern Blot analysis (Data not shown).

FIG. 22: Relative expression of LPA Receptors in HCT-8 cells. Data areexpressed as a fraction of TFIIB levels using earlier definedmethodologies.

FIG. 23: Ability of LPA to phosphorylate ERK in cell lines known toexpress OSGPR114. 10 μM LPA and 1 μM EGF were used for this experiment,and the cells were stimulated with ligand for 3 mins prior to lysing.

FIG. 24: Western blot showing the ability of LPA to transactivate EGFRin HCT-8 cells, as assessed using a phospho-tyrosine specific antibody.

FIG. 25. LPA (10 uM) activation of Shc, paxillin and SHP-2 in HCT-8 celllysates. Phospho-specific antibodies were used to detect the activatedproteins.

FIG. 26. Effect of LPA (10 μM), EGF (1 μM) and DMSO on levels ofphospho-Akt as a in a CHO-K1 cell line stably transfected with OSGPR114.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the identification of LPA, including, but notlimited to myristoyl lysophosphatidic acid, oleoyl lysophosphatidicacid, palmitoyl lysophosphatidic acid, and stearoyl lysophosphatidicacid, or a functional analog or homolog of one of these compounds, asligands of the G-protein coupled receptor OSGPR114 or OSGPR78, and isdirected to in vitro methods for screening candidate drugs for theirability to modulate the activity of OSGPR114 or OSGPR78, and to methodsof treating disease by administering to an individual a therapeuticamount of a modulator of OSGPR114 or OSGPR78.

The discovery that OSGPR114 or OSGPR78 is a physiological target proteinof LPA, a compound known to stimulate proliferation of many cell types,in addition to other activities, will allow for the identification anddevelopment of new drugs that also act via this protein, and for theidentification of other targets in the OSGPR114 or OSGPR78 signaltransduction pathways that can themselves be a target for new drugs.This invention is the first demonstration of an activation of theOSGPR114 or OSGPR78 receptor by LPA. Several physiological targetproteins for LPA have been described previously, but the ability of LPAto act as a ligand for the GPCR proteins OSGPR114 or OSGPR78 was unknownuntil the discovery described herein. Without knowledge of the targetprotein for compounds such as LPA it is very difficult and expensive todevelop effective compounds with either similar or antagonisticactivity. By providing alternative targets that LPA can act through, theinvention described herein provides a new approach to the development ofsuch compounds. Although one could have previously designed screensusing recombinant OSGPR114 and OSGPR78, there would have been littlemotivation to do so, as there was no indication that such compoundscould be used to modulate physiological and pathological processes thatinvolve LPA signaling, such as tumor growth. Furthermore, without theknowledge that LPA is the activating ligand for OSGPR114 and OSGPR78,the development of antagonists, or other modulators that inhibit theactivity of these receptors, would have been an extremely difficulttask.

This invention thus facilitates the design of simple and relativelyinexpensive screens to identify novel compounds that act via OSGPR114 orOSGPR78, and that have potential as modulators of cell growth and asanti-cancer agents. Furthermore, the recognition that OSGPR114 orOSGPR78 is a potential target for anti-cancer drugs will lead to theidentification of other proteins in the OSGPR114 or OSGPR78 signaltransduction pathway that can themselves be targets for potentialanti-cancer agents.

Prior to this invention, the utility of the OSGPR114 or OSGPR78 receptorwas unknown. The discovery that LPA, including myristoyllysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid, can act asligands for these GPCR proteins indicates that diseases or conditionsmediated by OSGPR114 or OSGPR78 include cancer (including, but notlimited to, ovarian cancer and other gynecological cancers (e.g.endometrial, cervical), cancers of the lung, prostate, pancreas, colon,breast, esophagus, kidney and stomach, and glioma, multiple myeloma,lymphoma, leukemia and melanoma), obesity, certain diseases of thecardiovascular and respiratory systems, including artherosclerosis,restenosis and acetylcholine-induced airway hyperresponsiveness, woundhealing, osteoporosis, inflammation, reproductive function, abnormalimmune system regulation, abnormalities in neuronal growth, survival andsignaling, renal failure associated with renal ischemia, andadditionally those affected by or induced by obesity, includingdiabetes, dyslipidemia and cardiovascular disease associated withobesity, including atherosclerosis, arteriosclerosis, hypercholesteremiaand hypertriglyceridemia, type 2 diabetes, type 1 diabetes, insulinresistance, metabolic syndrome (syndrome X) and hyperlipidemia. By theterm OSGPR114 or OSGPR78-mediated disease, it is meant those diseases orconditions where the modulation of OSGPR114 or OSGPR78 by agonists orantagonists results in a beneficial modification of the disease state orcondition.

Of the above conditions and diseases, antagonists, inverse agonists,partial inverse agonists and allosteric or allotopic antagonists ofOSGPR114 should generally be useful for treating certain cancers (e.g.ovarian cancer and other gynecological cancers, cancers of the lung,prostate, pancreas, colon, breast, esophagus, kidney and stomach, andglioma, lymphoma, leukemia and melanoma), obesity, atherosclerosis,restenosis, renal ischemia, and certain reproductive disorders.Agonists, partial agonists and allosteric or allotopic agonists ofOSGPR114 should generally be useful for treating wound healing andimmune system disorders requiring activation of immune system cells(e.g. T-cells). Additionally, inhibition of OSGPR114 or OSGPR78 (e.g. byantagonists, inhibition of gene expression, inhibition of downstreamsignaling pathways) will be useful in other conditions in which LPA isimplicated as a causative factor. Similarly, in diseases where LPA maybe beneficial (e.g. neuron growth, obesity), specific activators ofOSGPR114 or OSGPR78 or their signaling pathways could provetherapeutically beneficial.

In the case of neuronal disorders, LPA has been shown to have bothproliferative and apoptotic effects depending on the cell type (e.g.Xiaoqin Y., et. al. (2002) Biochem. Biophys. Acta 1585:108-113). Thusthe type of compound required for therapy of neural cells will depend onthe cell type and whether activation of inhibition of growth is desired(e.g. growth inhibition would be desirable in cancers of the CNS, butgrowth stimulation may be desirable in neural diseases where CNSregeneration is required). Furthermore, in certain other cell typeswhere LPA is found to have an effect opposite to that normallyencountered for that cell type, due perhaps to a different G-proteincoupling mechanism, the type of modulator required to treat a conditionor disease may be the opposite of that normally required for that celltype. Modulators of OSGPR114 or OSGPR78 that affect OSGPR114 or OSGPR78oligomerization (e.g. homodimerization or heterodimerization) orinteraction with other GPCRs may also be useful for treating theconditions and diseases listed above.

In addition to the effects of LPA mentioned above, increased OSGPR114 orOSGPR78 expression in certain cancer types (e.g. lung, breast and colon)provides additional evidence of involvement of OSGPR114 or OSGPR78 incancers of these tissues. Thus, modulation of OSGPR114 or OSGPR78activity by antagonists of these LPA activated receptors may result in abeneficial modification of these disease states or conditions.

OSGPR114 or OSGPR78 may therefore be used as a screening target for theidentification and development of novel pharmaceutical agents for use inthe methods of the invention. A modulator of OSGPR114 or OSGPR78 may beidentified by contacting a cell expressing on its surface the receptorOSGPR114 or OSGPR78, said receptor being associated with a secondcomponent capable of providing a detectable signal in response to thebinding of an agent to said receptor, with an agent to be screened underconditions to permit binding to the receptor; and determining whetherthe agent binds to, and activates, or inhibits, the receptor, bydetecting the presence or absence of a signal generated from theinteraction of the compound with the receptor and thereby determiningwhether the test agent modulates OSGPR114 or OSGPR78 activity. This maybe carried out in the presence of a labeled or unlabeled ligand, e.g.LPA, including, but are not limited to, myristoyl lysophosphatidic acid,oleoyl lysophosphatidic acid, palmitoyl lysophosphatidic acid, andstearoyl lysophosphatidic acid, or a functional analog or homolog of oneof these compounds.

An agent, or pharmaceutical agent, that can be tested for activity onthe OSGPR114 or OSGPR78 receptor includes any chemical compound,including small molecules (<approx. 5000 Daltons molecular weight) andmacromolecules (e.g. a polypeptide or protein, nucleic acid,glycoprotein, complex carbohydrate, synthetic or natural polymer etc.).Thus, an agent may be selected from combinatorial libraries, definedchemical entities, peptide and peptide mimetics, oligonucleotides andnatural product libraries, and other entities such as display (e.g.phage display libraries) and antibody products. In one embodiment, thetest agent is an LPA, or a closely related compound. Thus an agent thatmodulates the activity of OSGPR114 or OSGPR78 can be any chemicalcompound that binds to and modulates the activity of OSGPR114 orOSGPR78. Such agents that modulate the activity of OSGPR114 or OSGPR78can additionally be test agents for use in further methods or processesto determine their effects on cells or subjects, including animal modelsor patients.

This invention thus provides a method for identifying agents thatmodulate the activity of the OSGPR114 or OSGPR78 receptor, whichcomprises determining whether the agent interacts with OSGPR114 orOSGPR78 in a preparation comprising OSGPR114 or OSGPR78 receptorprotein. The method may be carried out in combination with a ligand forOSGPR114 or OSGPR78, wherein the ligand is an LPA, or a functionalanalog or homolog of such a compound. In one embodiment thelysophosphatidic acid is selected from 1-myristoyl lysophosphatidicacid, 1-oleoyl lysophosphatidic acid, 1-palmitoyl lysophosphatidic acid,and 1-stearoyl lysophosphatidic acid. In an alternative embodiment, theligand may be a compound that activates LPA receptors other thanOSGPR114 or OSGPR78, such as the Edg receptors LPA1, LPA2, and LPA3,including, for example, but not limited to, those described in WO02/29001; US 2003/0027800 A1; U.S. Pat. No. 6,380,177; Hasegawa, Y., etal. (2003) J. Biol. Chem. 278(14):11962-9; Heise, C. E., et al. (2001)Mol. Pharmacol 60(6):1173-80; Hooks, S. B., et al. (2001) J Biol. Chem.276(7):4611-21; Hopper, D. W., et al. (1999) J. Med. Chem. 42(6):963-70;Tigyi, G. (2001) Mol Pharmacol. 60(6):1161-4; Yokoyama, K., et al.(2002). Biochim. Biophys. Acta 1582(1-3): 295-308; Gueguen, G., et al.(1999). Biochemistry 38(26): 8440-50; Lynch, K. R. and T. L. Macdonald(2002). Biochim. Biophys. Acta 1582(1-3): 289-94; Sardar, V. M., et al.(2002) Biochim. Biophys. Acta 1582: 309-307 and Virag, T., et al. (2003)Mol Pharmacol. 63(5): 1032-42.

Furthermore, in any of the methods, processes or screening assaysdescribed herein as embodiments of this invention for identifyingcompounds which bind to or are modulators of OSGPR114 or OSGPR78activity, where a ligand or compound known to bind to or activate theOSGPR114 or OSGPR78 receptor is included in the method, process or assay(e.g. a competitive binding assay, or an assay for identifyingantagonists), the ligand may be an LPA. In the context of this inventionLPA (lysophosphatidic acid) is defined as a compound comprising aglycerol moiety with a fatty acid group at the sn1 (or sn2) position, aphosphate group at the sn3 position and a hydroxyl group at the sn2 (orsn1) site (e.g. Formula 1, 1-fatty acyl-LPA). The fatty acid group mayvary in the carbon chain length from C14 (myristoyl) through C22(docosatetraenoyl), i.e. can be selected from carbon chain lengths ofC14, C15, C16, C17, C18, C19, C20, C21 and C22. The fatty acid may besaturated or unsaturated. In one embodiment, the degree of unsaturationmay vary from one to six carbon double bonds. For example, suitablefatty acid groups include, but are not limited to, palmitoyl (C16:0),linoleoyl (C18:2), oleoyl (C18:1), stearoyl (C18:0) and arachidonyl(C20:4). Included within the definition of LPA are compounds in whichthe fatty acid ester linkage is replaced by an alkyl ether or alkenylether linkage, at either the sn1 or sn2 position. (e.g. formula 2). Inthese alkyl or alkenyl ether compounds the carbon chain length may varyfrom C14 through C22, i.e. can be selected from carbon chain lengths ofC14, C15, C16, C17, C18, C19, C20, C21 and C22. In one embodiment, thedegree of unsaturation in the alkenyl compounds may vary from one to sixcarbon double bonds.

Thus in one embodiment of this invention lysophosphatidic acid isselected from myristoyl lysophosphatidic acid, oleoyl lysophosphatidicacid, palmitoyl lysophosphatidic acid, stearoyl lysophosphatidic acid,linoleoyl lysophosphatidic acid, linolenoyl lysophosphatidic acid,arachidonoyl lysophosphatidic acid, myristoyl lysophosphatidic acid,elaidoyl lysophosphatidic acid, palmitoleoyl lysophosphatidic acid,petroselinoyl lysophosphatidic acid, palmitvaccenoyl lysophosphatidicacid, vaccenoyl lysophosphatidic acid, erucoyl lysophosphatidic acid,brassidoyl lysophosphatidic acid, clupanodonoyl lysophosphatidic acid,eleostearoyl lysophosphatidic acid, behenoyl lysophosphatidic acid,pentadecanoyl lysophosphatidic acid, heptadecanoyl (i.e. margaroyl)lysophosphatidic acid, nonadecanoyl lysophosphatidic acid, andhenicosanoyl lysophosphatidic acid.

In an alternative embodiment the lysophosphatidic acid is selected from1-myristoyl lysophosphatidic acid, 1-oleoyl lysophosphatidic acid,1-palmitoyl lysophosphatidic acid, and 1-stearoyl lysophosphatidic acid.

In yet another embodiment, in any of the methods, processes or screeningassays described herein as embodiments of this invention for identifyingcompounds which bind to or are modulators of OSGPR114 or OSGPR78activity, where a ligand or compound known to bind to or activate theOSGPR114 or OSGPR78 receptor is included in the method, process or assay(e.g. a competitive binding assay, or an assay for identifyingantagonists), the ligand may be a homolog or analog of LPA as definedabove.

Furthermore, in an alternative embodiment, in any of the methods,processes or screening assays described herein as embodiments of thisinvention for identifying compounds which bind to or are modulators ofOSGPR114 or OSGPR78 activity, where a ligand or compound known to bindto or activate the OSGPR114 or OSGPR78 receptor is included in themethod, process or assay (e.g. a competitive binding assay, or an assayfor identifying antagonists), the ligand may be a compound thatactivates LPA receptors other than OSGPR114 or OSGPR78, such as the Edgreceptors LPA1, LPA2, and LPA3, including, for example, but not limitedto, those described in WO 02/29001; US 2003/0027800 A1; U.S. Pat. No.6,380,177 Hasegawa, Y., et al. (2003) J. Biol. Chem. 278(14):11962-9;Heise, C. E., et al. (2001) Mol. Pharmacol 60(6):1173-80; Hooks, S. B.,et al. (2001) J Biol. Chem. 276(7):4611-21; Hopper, D. W., et al. (1999)J. Med. Chem. 42(6):963-70; Tigyi, G. (2001) Mol Pharmacol.60(6):1161-4; Yokoyama, K., et al. (2002). Biochim. Biophys. Acta1582(1-3): 295-308; Gueguen, G., et al. (1999). Biochemistry 38(26):8440-50; Lynch, K. R. and T. L. Macdonald (2002). Biochim. Biophys. Acta1582(1-3): 289-94; Sardar, V. M., et al. (2002) Biochim. Biophys. Acta1582: 309-307 and Virag, T., et al. (2003) Mol Pharmacol. 63(5): 1032-42as compounds that are activators of LPA receptors (edg receptors). Theligand may be specific compounds as described therein, or specificexamples of structures that are encompassed by a structure that definesa genus.

Furthermore, in any of the methods, processes or screening assaysdescribed herein as embodiments of this invention for identifyingcompounds which bind to or are modulators of OSGPR114 or OSGPR78activity, where a ligand or compound known to bind to or activate theOSGPR114 or OSGPR78 receptor is included in the method, process orassay, in one embodiment a ligand concentration is selected thatproduces a submaximal activation of OSGPR114 or OSGPR78 (e.g. 5%, 10%,20%, or 50% of maximal response) in order to identify agonists, partialagonists or allosteric or allotopic agonists. Identification ofallosteric or allotopic agonists will be dependent on the presence ofsuch an activating ligand, and would not have been possible prior to theidentification of LPA as a ligand for OSGPR114 or OSGPR78 as describedherein.

For example, a method for identification of an agent that modulatesOSGPR114 or OSGPR78 comprises (i) contacting a test agent with a cell(including but not limited to cells such as a breast, colon, lung orovarian cell, or other cells known to express OSGPR114 or OSGPR78) whichexpresses OSGPR114 or OSGPR78 or a variant thereof that is capable ofcoupling to a G-protein; and (ii) monitoring for OSGPR114 or OSGPR78activity in the presence of a G protein; thereby determining whether thetest agent modulates OSGPR114 or OSGPR78 activity.

The test agent may be contacted in step (i) with cells that expressOSGPR114 or OSGPR78 or a variant thereof. Alternatively, the test agentmay be contacted in step (i) with membranes obtained from such cells. Inone embodiment, a modulator of OSGPR114 or OSGPR78 may be identified bydetermining the inhibition of binding of a ligand to cells which havethe receptor on the surface thereof, or to cell membranes containing thereceptor, in the presence of a candidate compound, under conditions topermit binding to the receptor, and determining the amount of ligandbound to the receptor, such that a compound capable of causing reductionof binding of a ligand is an agonist or antagonist, in which method theligand is an LPA compound, including, but not limited to, myristoyllysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid, or afunctional analog or homolog of one of these compounds.

This invention thus provides a method of identifying a modulator ofOSGPR114 or OSGPR78 activity, comprising (a) providing a OSGPR114 orOSGPR78 receptor, (b) incubating the OSGPR114 or OSGPR78 receptor withan test agent to be screened under conditions to permit binding of thetest agent to the receptor; and (c) determining whether the test agentbinds to, and activates, or inhibits, the receptor, by detecting thepresence or absence of a signal generated from the interaction of theagent with the receptor, and thereby determining whether the test agentmodulates OSGPR114 or OSGPR78 activity.

This invention thus also provides a method of identifying a modulator ofOSGPR114 or OSGPR78, comprising (a) providing a cell expressing on itssurface the receptor OSGPR114 or OSGPR78, said receptor being associatedwith a second component capable of providing a detectable signal inresponse to the binding of an agent to said receptor, (b) contactingwith an test agent to be screened under conditions to permit binding tothe receptor; and (c) determining whether the agent binds to, andactivates, or inhibits, the receptor, by detecting the presence orabsence of a signal generated from the interaction of the compound withthe receptor and thereby determining whether the test agent modulatesOSGPR114 or OSGPR78 activity.

This invention further provides the above methods wherein the step ofincubating or contacting with the test agent (step b) is carried out incombination with (i.e. in the presence of; or by also contacting thecells with) a ligand for the OSGPR114 or OSGPR78 receptor, in whichmethod the ligand is an LPA, including, but not limited to, myristoyllysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid, or afunctional analog or homolog of one of these compounds.

In the practice of this invention the OSGPR114 or OSGPR78 receptor canbe from any species, or a functional variant thereof, as describedherein. In one embodiment, the OSGPR114 or OSGPR78 receptor is selectedfrom human, mammalian, rodent, murine, rat, dog, rabbit and monkeyreceptors. The cells of this invention can be any cells expressingOSGPR114 or OSGPR78 receptor or a functional variant thereof. Suitablecells are colon, lung, ovarian, breast, prostate, pancreas, esophagus,kidney, stomach, glioma, leukocyte, lymphocyte, melanocyte, andadipocyte cells, or cells where the OSGPR114 or OSGPR78 receptorexpressed by the cell is a recombinant receptor, including CHO, CHO-K1,RH7777, Jurkat, HCT4, RBL243, HeLa, ASPC-1, HEK-293, and COS7 cells.

Cells can be provided as a differentiated cell line, or can be primarycells harvested from a human or animal donor. In the methods of thisinvention, where the OSGPR114 or OSGPR78 is associated with a secondcomponent capable of providing a detectable signal, that secondcomponent may be a G-protein, for example a Gi or a Go-protein, or aG-protein with a promiscuous G-alpha subunit (e.g. G₁₆, G₁₅). In analternative embodiment that second component may be a Gs, Gq or G₁₂₋₁₃protein. In a further embodiment the second component can be β-arrestin.In one embodiment, where OSGPR114 is associated with a second componentcapable of providing a detectable signal, that second component is aGi-protein or a Go-protein. In one embodiment, where OSGPR78 isassociated with a second component capable of providing a detectablesignal, that second component is a Gs-protein.

The invention also provides a test kit suitable for identification of anagent that modulates OSGPR114 or OSGPR78 activity, which kit comprises(a) OSGPR114 or OSGPR78 or a variant thereof which is capable ofcoupling to a G-protein; and (b) means for monitoring OSGPR114 orOSGPR78 activity. The G-protein may be selected from Gi, Go, G-₁₆, G-₁₅,Gs, Gq and G₁₂₋₁₃, or any other G-protein that is activated by OSGPR114or OSGPR78 under the conditions employed in the test kit. In oneembodiment, component (a) comprises cells which express OSGPR114 orOSGPR78, or a variant thereof. In one embodiment the means formonitoring OSGPR114 or OSGPR78 activity of component (b) is an assaysystem for monitoring a signal transduction pathway or cellular activityactivated by the coupling of OSGPR114 or OSGPR78 or a variant thereof toa G-protein. Several such assay systems are described herein, or arewell known by one of ordinary skill in the art. In one embodimentcomponent (b) comprises a means for determining whether Gi/o isactivated.

This invention also provides a method for identification of an agentthat modulates a cellular activity regulated by activation of OSGPR114or OSGPR78 in a cell, which method comprises contacting a test cell witha test agent that modulates OSGPR114 or OSGPR78 activity, and which hasbeen identified by the method of the invention (e.g. an assay method orprocess described herein), monitoring a change in the cellular activity,and thereby determining whether the test substance is a modulator of thecellular activity. This method can also be performed where the step ofcontacting a test cell with a test agent is carried out in combinationwith a ligand for the OSGPR114 or OSGPR78 receptor. The inventionfurther provides this method but wherein the test agent is not known tomodulate OSGPR114 or OSGPR78 activity, and wherein the step ofcontacting a test cell with a test agent is carried out in combinationwith a ligand for the OSGPR114 or OSGPR78 receptor. In these methods theligand is an LPA compound, including, but not limited to, myristoyllysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid, or afunctional analog or homolog of one of these compounds. Competition ofthe test agent with the ligand will identify agents (e.g. antagonists,reverse agonists) that can modulate regulation of the cellular activityby the ligand.

In the preceding methods for identification of an agent that modulates acellular activity regulated by activation of OSGPR114 or OSGPR78 in acell, the cellular activity can be, but is not limited to, cellproliferation, apoptosis, anoikis (in endothelial or epithelial cells),proangiogenic factor secretion, cell invasion, cell motility,chemotaxis, insulin secretion, ion flux across cell membranes (e.g.Ca²⁺, K⁺, Na⁺), cellular aggregation, closure of gap junctions, neuriteretraction, changes in cytoskeletal architecture, ERK activation, c-fosgene induction, cellular LPA production, metalloproteinase activity(e.g. MMP2, MMP9), urokinase plasminogen activator (uPA) production,urokinase plasminogen activator activity, lipid metabolism, storage ortransport, and glucose metabolism or transport. In one embodiment thecell is a colon, lung, ovarian, breast, prostate, pancreas, esophagus,kidney, stomach, glioma, leukocyte, lymphocyte, or melanocyte cell. Inan alternative embodiment the cell is an adipocyte, a pancreatic cell, arenal cell, a hepatocyte, or a skeletal muscle cell.

This invention also provides a method for identification of an agentthat inhibits tumor growth, which method comprises contacting a testsubject with a test agent which modulates OSGPR114 or OSGPR78 activity,and monitoring tumor growth, thereby determining whether the testsubstance is an inhibitor of tumor growth.

This invention also provides a method for identification of an agentthat inhibits tumor growth, which method comprises contacting a testsubject with a test agent which inhibits LPA-activated OSGPR114 orOSGPR78 activity, and monitoring tumor growth or tumor size, therebydetermining whether the test substance is an inhibitor of tumor growth.

This invention also provides a method for identification of an agentthat inhibits cell proliferation, which method comprises contacting acell with a test agent which modulates OSGPR114 or OSGPR78 activity, andmonitoring cell proliferation, thereby determining whether the testsubstance is an inhibitor of cell proliferation.

This invention also provides a method for identification of an agentthat inhibits cell proliferation, which method comprises contacting acell with a test agent which inhibits LPA-activated OSGPR114 or OSGPR78activity, and monitoring cell proliferation, thereby determining whetherthe test substance is an inhibitor of cell proliferation.

This invention also provides a method for identification of an agentthat stimulates cell proliferation, which method comprises contacting acell with a test agent which modulates OSGPR114 or OSGPR78 activity, andmonitoring cell proliferation, thereby determining whether the testsubstance is a stimulator of cell proliferation.

This invention also provides a method for identification of an agentthat stimulates cell proliferation, which method comprises contacting acell with a test agent which further activates or enhances LPA-activatedOSGPR114 or OSGPR78 activity, and monitoring cell proliferation, therebydetermining whether the test substance is a stimulator of cellproliferation. An example of a test substance that would furtheractivate or enhance LPA-activated OSGPR114 or OSGPR78 activity would bean allosteric agonist.

This invention also provides a method for identification of an agentthat inhibits tumor growth, which method comprises contacting a testsubject with a test agent which modulates OSGPR114 or OSGPR78 activity,and which has been identified by any method or process of the invention,and monitoring tumor growth or tumor size, thereby determining whetherthe test substance is an inhibitor of tumor growth.

This invention also provides a method for identification of an agentthat inhibits tumor growth, which method comprises contacting a testsubject with a test agent which inhibits LPA-activated OSGPR114 orOSGPR78 activity, and which has been identified by any method or processof the invention, and monitoring tumor growth or tumor size, therebydetermining whether the test substance is an inhibitor of tumor growth.

This invention also provides a method for identification of an agentthat inhibits cell proliferation, which method comprises contacting acell with a test agent which modulates OSGPR114 or OSGPR78 activity, andwhich has been identified by any method or process of the invention, andmonitoring cell proliferation, thereby determining whether the testsubstance is an inhibitor of cell proliferation.

This invention also provides a method for identification of an agentthat inhibits cell proliferation, which method comprises contacting acell with a test agent which inhibits LPA-activated OSGPR114 or OSGPR78activity, and which has been identified by any method or process of theinvention, and monitoring cell proliferation, thereby determiningwhether the test substance is an inhibitor of cell proliferation.

This invention also provides a method for identification of an agentthat stimulates cell proliferation, which method comprises contacting acell with a test agent which modulates OSGPR114 or OSGPR78 activity, andwhich has been identified by any method or process of the invention, andmonitoring cell proliferation, thereby determining whether the testsubstance is a stimulator of cell proliferation.

This invention also provides a method for identification of an agentthat stimulates cell proliferation, which method comprises contacting acell with a test agent which further activates or enhances LPA-activatedOSGPR114 or OSGPR78 activity, and which has been identified by anymethod or process of the invention, and monitoring cell proliferation,thereby determining whether the test substance is a stimulator of cellproliferation. An example of a test substance that would furtheractivate or enhance LPA-activated OSGPR114 or OSGPR78 activity would bean allosteric agonist.

This invention also provides a method for identification of an agentthat causes an activation of cell apoptosis, which method comprisescontacting a cell with a test agent which modulates OSGPR114 or OSGPR78activity, identified by any method or process of the invention, andmonitoring cell apoptosis, thereby determining whether the test agent isan activator of apoptosis.

This invention also provides a method for identification of an agentthat causes an inhibition of cell apoptosis, which method comprisescontacting a cell with a test agent which modulates OSGPR114 or OSGPR78activity, identified by any method or process of the invention, andmonitoring cell apoptosis, thereby determining whether the test agent isan inhibitor of apoptosis.

This invention also provides a method for identification of an agentthat causes a reduction in cell motility, which method comprisescontacting a cell with a test agent which modulates OSGPR114 or OSGPR78activity, identified by any method or process of the invention, andmonitoring cell motility, thereby determining whether the test agent isan inhibitor of cell motility.

This invention also provides a method for identification of an agentthat causes an increase in cell motility, which method comprisescontacting a cell with a test agent which modulates OSGPR114 or OSGPR78activity, identified by any method or process of the invention, andmonitoring cell motility, thereby determining whether the test agent isan activator of cell motility.

This invention also provides a method for identification of an agentthat causes a reduction in cell invasion, which method comprisescontacting a cell with a test agent which modulates OSGPR114 or OSGPR78activity, identified by any method or process of the invention, andmonitoring cell invasion, thereby determining whether the test agent isan inhibitor of cell invasion.

This invention also provides a method for identification of an agentthat causes an increase in cell invasion, which method comprisescontacting a cell with a test agent which modulates OSGPR114 or OSGPR78activity, identified by any method or process of the invention, andmonitoring cell invasion, thereby determining whether the test agent isan stimulator of cell invasion.

This invention also provides a method for identification of an agentthat causes an increase in proangiogenic factor secretion from cells,which method comprises contacting a cell with a test agent whichmodulates OSGPR114 or OSGPR78 activity, identified by any method orprocess of the invention, and monitoring proangiogenic factor secretion,thereby determining whether the test agent is an activator ofproangiogenic factor secretion.

This invention also provides a method for identification of an agentthat causes a decrease in proangiogenic factor secretion from cells,which method comprises contacting a cell with a test agent whichmodulates OSGPR114 or OSGPR78 activity, identified by any method orprocess of the invention, and monitoring proangiogenic factor secretion,thereby determining whether the test agent is an inhibitor ofproangiogenic factor secretion.

This invention also provides a method for identification of an agentthat causes an activation of cell apoptosis in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring cell apoptosis, thereby determining whetherthe test agent is an activator of apoptosis.

This invention also provides a method for identification of an agentthat causes an inhibition of cell apoptosis in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring cell apoptosis, thereby determining whetherthe test agent is an inhibitor of apoptosis.

This invention also provides a method for identification of an agentthat causes a reduction in cell motility in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring cell motility, thereby determining whether thetest agent is an inhibitor of cell motility.

This invention also provides a method for identification of an agentthat causes a increase in cell motility in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring cell motility, thereby determining whether thetest agent is an activator of cell motility.

This invention also provides a method for identification of an agentthat causes a reduction in cell invasion in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring cell invasion, thereby determining whether thetest agent is an inhibitor of cell invasion.

This invention also provides a method for identification of an agentthat causes a increase in cell invasion in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring cell invasion, thereby determining whether thetest agent is an stimulator of cell invasion.

This invention also provides a method for identification of an agentthat causes a increase in proangiogenic factor secretion from cells in asubject, which method comprises contacting a subject with a test agentwhich modulates OSGPR114 or OSGPR78 activity, identified by any methodor process of the invention, and monitoring proangiogenic factorsecretion, thereby determining whether the test agent is an activator ofproangiogenic factor secretion.

This invention also provides a method for identification of an agentthat causes a decrease in proangiogenic factor secretion from cells in asubject, which method comprises contacting a subject with a test agentwhich modulates OSGPR114 or OSGPR78 activity, identified by any methodor process of the invention, and monitoring proangiogenic factorsecretion, thereby determining whether the test agent is an inhibitor ofproangiogenic factor secretion.

This invention also provides a method for identification of an agentthat causes a reduction in angiogenesis in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring angiogenesis, thereby determining whether thetest agent is an inhibitor of angiogenesis.

This invention also provides a method for identification of an agentthat causes a increase in angiogenesis in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring angiogenesis, thereby determining whether thetest agent is an activator of angiogenesis.

This invention also provides a method for identification of an agentthat causes a reduction in metastasis in a subject, which methodcomprises contacting a subject with a test agent which modulatesOSGPR114 or OSGPR78 activity, identified by any method or process of theinvention, and monitoring metastasis, thereby determining whether thetest agent is an inhibitor of metastasis.

In all the preceding methods for identification of agents, the testagent can be an antagonist, an inverse agonist, a partial inverseagonist, an allosteric or allotopic antagonist, an agonist, a partialagonist, or an allosteric or allotopic agonist.

This invention also provides any of the above methods involvingcontacting a cell, wherein the cell is a colon, lung, ovarian, breast,prostate, pancreas, esophagus, kidney, stomach, glioma, leukocyte,lymphocyte, melanocyte, or adipocyte cell.

This invention also provides any of the above methods for identificationof an agent that inhibits tumor growth, wherein the tumor is a colon,lung, cervical, ovarian, endometrial, breast, prostate, pancreas,esophagus, kidney, stomach, glioma, myeloma or melanoma tumor.

This invention also provides a modulator of OSGPR114 or OSGPR78activity, or a modulator of cell proliferation or tumor growth,identified by a method of the invention, and their use in therapy andpharmaceutical compositions comprising them.

This invention also provides a method of inhibiting tumor growth in amammal in recognized need of such treatment, said method comprisingadministering to said mammal in recognized need of such treatment, anantagonist or inverse agonist of OSGPR114 or OSGPR78 activity, whereinsaid administering is in an effective amount to inhibit tumor growth insaid mammal.

This invention also provides a method of inhibiting tumor growth in amammal in recognized need of such treatment, said method comprisingadministering to said mammal in recognized need of such treatment, anantagonist of the LPA-dependent or LPA-activated activity of OSGPR114 orOSGPR78, wherein said administering is in an effective amount to inhibittumor growth in said mammal.

In the above methods of inhibiting tumor growth, the antagonist may be asmall molecule that competes with the LPA ligand on the OSGPR114 orOSGPR78 receptor. Alternatively, the antagonist may be an agent thatreduces the level of OSGPR114 or OSGPR78 receptor protein in the tumorcells, e.g. an antisense molecule, or a small inhibitory RNA (i.e. RNAinterference). Methods for designing and using effective molecules ofthis type for genes whose sequence is known are well known in the art,as described in references cited herein (see p. 33-34).

This invention also provides a method of inhibiting tumor growth in amammal in recognized need of such treatment, said method comprisingadministering to said mammal in recognized need of such treatment, anagonist or allosteric agonist of an LPA-activated mammalian OSGPR114 orOSGPR78 receptor, wherein said administering is in an effective amountto reduce tumor growth in said mammal.

In one embodiment of the preceding methods the tumor is a colon, lung,cervical, ovarian, endometrial, breast, prostate, pancreas, esophagus,kidney, stomach, glioma, myeloma or melanoma tumor.

This invention also provides a method of reducing abnormal cell growthor abnormal cell proliferation in a mammal in recognized need of suchtreatment, said method comprising administering to said mammal inrecognized need of such treatment, an antagonist of an LPA-activatedmammalian OSGPR114 or OSGPR78 receptor, wherein said administering is inan effective amount to reduce abnormal cell growth in said mammal. Inone embodiment of this method the cell is a colon, lung, ovarian,breast, prostate, cervical, pancreas, esophagus, kidney, stomach,glioma, leukocyte, lymphocyte, or melanocyte cell.

“Abnormal cell growth” or “abnormal cell proliferation”, as used herein,refers to cell growth that is independent of normal regulatorymechanisms (e.g. as relected by loss of contact inhibition of cellsgrown in culture in vitro), including the abnormal growth of normalcells and the growth of abnormal cells. This includes, but is notlimited to, the abnormal growth of tumor cells (tumors), both benign andmalignant, and the growth of cells in diseases associated with benigncell proliferation, e.g. atherosclerosis, restenosis.

This invention also provides a method of stimulating apoptosis of cellsin a mammal in recognized need of such treatment, said method comprisingadministering to said mammal in recognized need of such treatment, anantagonist of an LPA-activated mammalian OSGPR114 or OSGPR78 receptor,wherein said administering is in an effective amount to stimulateapoptosis in said mammal. In one embodiment of this method the cells arecolon, lung, ovarian, breast, prostate, pancreas, esophagus, kidney,stomach, glioma, leukocyte, lymphocyte, or melanocyte cells.

This invention also provides a method of inhibiting angiogenesis in amammal in recognized need of such treatment, said method comprisingadministering to said mammal in recognized need of such treatment, anantagonist of an LPA-activated mammalian OSGPR114 or OSGPR78 receptor,wherein said administering is in an effective amount to inhibitangiogenesis in said mammal.

This invention also provides a method of stimulating angiogenesis in amammal in recognized need of such treatment, said method comprisingadministering to said mammal in recognized need of such treatment, anagonist of a mammalian OSGPR114 or OSGPR78 receptor, wherein saidadministering is in an effective amount to stimulate angiogenesis insaid mammal.

This invention also provides a method of inhibiting metastasis in amammal in recognized need of such treatment, said method comprisingadministering to said mammal in recognized need of such treatment, anantagonist of an LPA-activated mammalian OSGPR114 or OSGPR78 receptor,wherein said administering is in an effective amount to inhibitmetastasis in said mammal.

In the above methods of reducing abnormal cell growth or abnormal cellproliferation, stimulating angiogenesis, or inhibiting metastasis, theantagonist may be a small molecule that competes with the LPA ligand onthe OSGPR114 or OSGPR78 receptor. Alternatively, the antagonist may bean agent that reduces the level of OSGPR114 or OSGPR78 receptor proteinin the tumor cells, e.g. an antisense molecule, or a small inhibitoryRNA (i.e. RNA interference). Methods for designing and using effectivemolecules of this type for genes whose sequence is known are well knownin the art, as described in references cited herein (see p. 33-34).

This invention also provides a modulator (e.g. an activator or aninhibitor) of OSGPR114 or OSGPR78 activity, or a modulator (e.g. anactivator or an inhibitor) of cell proliferation or tumor growth,identified by a method of the invention, or a polynucleotide whichencodes OSGPR114 or OSGPR78 or a variant polypeptide, for use in amethod of treatment of the human or animal body by therapy; and use ofsuch a modulator (e.g. activator, inhibitor) or polynucleotide in themanufacture of a medicament for the treatment of diseases or conditionsmodulated by OSGPR114 or OSGPR78, for example, cancer, obesity, certaindiseases of the cardiovascular and respiratory systems, includingartherosclerosis, restenosis and acetylcholine induced airwayhyperresponsiveness, wound healing, reproductive function, abnormalimmune system regulation, abnormalities in neuronal growth, survival andsignaling, and renal failure associated with renal ischemia.

This invention also provides an inhibitor of cell proliferation,apoptosis, cell motility, cell invasion, proangiogenic factor secretion,angiogenesis, tumor growth, or metastasis identified by a method of thisinvention. This invention further provides the use of the inhibitor inthe manufacture of a medicament for the treatment of abnormal cellproliferation or cancer. In one embodiment the inhibitor ofproliferation, apoptosis, cell motility, cell invasion, proangiogenicfactor secretion, angiogenesis, tumor growth, or metastasis is for usein the treatment of abnormal cell proliferation or cancer. In oneembodiment the abnormal cell proliferation or cancer is selected fromcancers of the colon, lung, ovarian, breast, prostate, pancreas,esophagus, kidney, and stomach, and glioma, leukemia, lymphoma, andmelanoma.

This invention also provides an activator of cell proliferation,apoptosis, cell motility, cell invasion, proangiogenic factor secretion,or angiogenesis identified by a method of this invention. This inventionfurther provides the use of the activator in the manufacture of amedicament for the treatment of abnormal cell proliferation or cancer.In one embodiment the activator of apoptosis is for use in the treatmentof abnormal cell proliferation or cancer. In one embodiment the abnormalcell proliferation or cancer is selected from cancers of the colon,lung, ovarian, breast, prostate, pancreas, esophagus, kidney, andstomach, and glioma, leukemia, lymphoma, and melanoma.

This invention further provides the use of a modulator of OSGPR114 orOSGPR78 for the manufacture of a medicament for the treatment ofOSGPR114 or OSGPR78 mediated diseases. In one embodiment the OSGPR114 orOSGPR78 mediated disease is selected from cancers of the colon, lung,ovarian, breast, prostate, pancreas, esophagus, kidney, and stomach, andglioma, leukemia, lymphoma, and melanoma.

The invention also provides a method of treating OSGPR114 or OSGPR78mediated disease in an individual which comprises administering to theindividual a therapeutic amount of a modulator of OSGPR114 or OSGPR78activity. In one embodiment the OSGPR114 or OSGPR78 mediated disease isselected from cancers of the colon, lung, ovarian, breast, prostate,pancreas, esophagus, kidney, and stomach, and glioma, leukemia,lymphoma, and melanoma.

This invention further provides a method of treating an OSGPR114 orOSGPR78 mediated disease in an individual which comprises administeringto the individual a therapeutic amount of a modulator of OSGPR114 incombination with a therapeutic amount of a modulator of OSGPR78activity. In one embodiment the OSGPR114 or OSGPR78 mediated disease isselected from cancers of the colon, lung, ovarian, breast, prostate,pancreas, esophagus, kidney, and stomach, and glioma, leukemia,lymphoma, and melanoma.

This invention provides a method for inhibiting tumor growth that ismodulated by the LPA-activated activity of the OSGPR114 or OSGPR78receptor, the method comprising reducing the LPA-activated activity ofOSGPR114 or OSGPR78 in the tumor such that tumor growth is inhibited.

In one embodiment of this method for inhibiting tumor growth, the tumoris selected from a colon, lung, ovarian, breast, prostate, pancreas,esophagus, kidney, stomach, glioma, or melanoma tumor.

This invention provides a method for inhibiting angiogenesis in asubject that is modulated by the LPA-activated activity of the OSGPR114or OSGPR78 receptor, the method comprising reducing the LPA-activatedactivity of OSGPR114 or OSGPR78 in the subject such that angiogenesis isinhibited.

This invention provides a method for inhibiting metastasis in a subjectthat is modulated by the LPA-activated activity of the OSGPR114 orOSGPR78 receptor, the method comprising reducing the LPA-activatedactivity of OSGPR114 or OSGPR78 in the subject such that metastasis isinhibited.

This invention provides a method for inhibiting a cellular activity thatis modulated by the LPA-activated activity of the OSGPR114 or OSGPR78receptor, the method comprising reducing the LPA-activated activity ofOSGPR114 or OSGPR78 in the cell such that the cellular activity isinhibited. The cellular activity includes, but is not limited to, cellproliferation, apoptosis, cell motility, cell invasion, andproangiogenic factor secretion.

In one embodiment of the above methods wherein the LPA-activatedactivity of OSGPR114 or OSGPR78 activity is reduced, the activity isreduced by contacting the tumor, subject or cell with a small moleculeinhibitor (e.g. an antagonist) of the LPA-activated activity of OSGPR114or OSGPR78. In another embodiment of the above methods wherein theLPA-activated activity of OSGPR114 or OSGPR78 activity is reduced, theactivity is reduced by specifically inhibiting transcription or geneexpression from the OSGPR114 or OSGPR78 gene. In one embodiment, geneexpression can be reduced by contacting the tumor, subject or cell witha double stranded RNA (dsRNA), or a vector or construct causing theproduction of double stranded RNA, such that expression of the OSGPR114or OSGPR78 receptor is specifically inhibited (i.e. RNA interference orRNAi). Methods for selecting an appropriate dsRNA or dsRNA-encodingvector are well known in the art for genes whose sequence is known (e.g.see Tuschi, T., et al. (1999) Genes Dev. 13(24):3191-3197; Elbashir, S.M. et al. (2001) Nature 411:494-498; Hannon, G. J. (2002) Nature418:244-251; McManus, M. T. and Sharp, P. A. (2002) Nature ReviewsGenetics 3:737-747; Bremmelkamp, T. R. et al. (2002) Science296:550-553; U.S. Pat. No. 6,573,099; WO 01/36646; WO 99/32619; U.S.Pat. No. 6,506,559; WO 01/68836). In another embodiment, gene expressioncan be reduced by contacting the tumor, subject or cell with a specificsmall molecule inhibitor of transcription. Methods for identifyingcompounds that are specific modulators of transcription are well knownin the art (e.g. U.S. Pat. No. 5,776,502, U.S. Pat. No. 5,665,543). Inanother embodiment, gene expression can be reduced by contacting thetumor, subject or cell with an antisense molecule. Methods for usingantisense techniques for specifically inhibiting gene expression ofgenes whose sequence is known are also well known in the art (e.g. seeU.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091;6,046,321; and 5,981,732).

This invention provides a method for stimulating a cellular activitythat is modulated by the LPA-activated activity of the OSGPR114 orOSGPR78 receptor, the method comprising increasing the LPA-activatedactivity of OSGPR114 or OSGPR78 in the cell such that the cellularactivity is stimulated. The cellular activity includes, but is notlimited to, cell proliferation, apoptosis, cell motility, cell invasion,and proangiogenic factor secretion.

In one embodiment of the above methods wherein the LPA-activatedactivity of OSGPR114 or OSGPR78 activity is stimulated, the activity isincreased by contacting the tumor, subject or cell with a small moleculeinhibitor (e.g. an agonist, an allosteric agonist) of the LPA-activatedactivity of OSGPR114 or OSGPR78, or the activity is increased byspecifically stimulating transcription from the OSGPR114 or OSGPR78gene.

In one embodiment of the above methods for stimulating a cellularactivity that is modulated by the LPA-activated activity of the OSGPR114or OSGPR78 receptor, the cell is a colon, lung, ovarian, breast,prostate, pancreas, esophagus, kidney, stomach, glioma, leukocyte,lymphocyte, or melanocyte cell. In an alternative embodiment the cell isan adipocyte, a pancreatic cell, a renal cell, a hepatocyte, or askeletal muscle cell.

The present invention relates to the use of the human G-protein coupledreceptor OSGPR114 or OSGPR78, and variants thereof. Human and mouseOSGPR114 or OSGPR78 have been cloned previously. Human OSGPR114 andOSGPR78 receptor encoding DNA have the GenBank Accession numbersAJ272207 and AF000546 respectively. Mouse OSGPR114 and OSGPR78 receptorencoding DNA have the GenBank Accession numbers AY255621 andNM_(—)175116 respectively. The term OSGPR114 or OSGPR78 as used hereinincorporates variants of OSGPR114 or OSGPR78. OSGPR114 or OSGPR78receptors for use in the screening methods of the invention include allspecies orthologues, e.g., may be mammalian, rodent, mouse, rat, rabbit,dog, monkey or human. Human nucleic acid and amino acid sequences aredepicted in FIGS. 1-2. Human OSGPR114 or OSGPR78 is preferred. The term“variant” refers to a polypeptide which has the same essential characteror basic biological functionality as OSGPR114 or OSGPR78. The essentialcharacter of OSGPR114 or OSGPR78 can be defined as that of a G-proteincoupled receptor of similar structure that is activated by LPA asdescribed herein. Thus, the term “variant” refers with respect toOSGPR114 in particular to a GPCR polypeptide of similar structure thatactivates a Gi/o G-protein response in response to LPA under theexperimental conditions described herein (e.g. see FIG. 10).

To determine whether a candidate variant has the same function asOSGPR114 or OSGPR78, the ability of the variant to activate Gi/o-proteinor other G-proteins can be determined. The effect of the candidatevariant on Gi/o activation can be monitored. This can be carried out,for example, by contacting cells expressing the candidate variant with aligand which activates Gi/o-protein when contacted with cells thatexpress OSGPR114 or OSGPR78, and measuring a Gi/o-coupled readout. Acontrol experiment is typically also carried out in which cells of thesame type as those expressing the candidate variant, but expressingOSGPR114 or OSGPR78 instead, are contacted with the ligand and acorresponding Gi/o-coupled readout is measured. The effect attained bythe candidate variant can then be directly compared with that attainedby OSGPR114 or OSGPR78. It should be noted that, although OSGPR114 orOSGPR78 may activate a Gi/o-protein or other G-protein response underthe experimental conditions employed herein (e.g. see FIG. 10 forOSGPR114 activation), under different experimental conditions OSGPR114or OSGPR78 may activate other G-proteins. Thus, when differentexperimental conditions are employed the G-protein activated by OSGPR114or OSGPR78 may have to be re-determined prior to comparison with acandidate variant.

Alternatively, a variant polypeptide is one of similar structure whichbinds to the same ligand as OSGPR114 or OSGPR78. That can be determineddirectly by contacting a candidate variant with a radiolabelled ligandthat binds to OSGPR114 or OSGPR78 and monitoring binding of the ligandto the variant. Typically, the radiolabelled ligand can be incubatedwith cell membranes containing the candidate variant. The membranes canthen be separated from non-bound ligand and dissolved in scintillationfluid to allow the radioactivity of the membranes to be determined byscintillation counting. Non-specific binding of the candidate variantmay also be determined by repeating the experiment in the presence of asaturating concentration of non-radioactive ligand. Preferably a bindingcurve is constructed by repeating the experiment with variousconcentrations of the candidate variant. The ability of OSGPR114 orOSGPR78 to bind a ligand may also be determined indirectly as describedbelow. Surface plasmon resonance methodology can also be utilized, withthe advantage that radiolabelling is not required (e.g. see technologyreviews as published by Biacore, in print and on their website; Myszka,D. G. and Rich, R. L. (2000) Pharmaceutical Science and TechnologyToday, 3:310-317; Quinn, J. G. et. al. (2000) Anal. Biochem.281:135-143; Williams, C. (2000) Current Opinion Biotech. 11:42-46).Additional methods of determining and characterizing ligand binding arewell known in the art (e.g. see Kenakin, T. (1997) MolecularPharmacology, A Short Course. p. 1-235, Blackwell Science).

Typically, polypeptides with more than about 65% identity, preferably atleast 80% or at least 90% and particularly preferably at least 95%, atleast 97% or at least 99% identity, with the amino acid sequences ofhuman or mouse OSGPR114 or OSGPR78 sequences as described in the Genbankdatabase, or more preferably over a region of at least 20, preferably atleast 30, at least 40, at least 60 or at least 100 contiguous aminoacids or over the full length of the amino acid sequences are consideredas OSGPR114 or OSGPR78 variants. The UWGCG Package provides the BESTFITprogram which can be used to calculate identity (for example used on itsdefault settings) (Devereau et al (1984) Nucleic Acid Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculateidentity or line up sequences (typically on their default settings), forexample as described in Altschul S. F. (1993) J. Mol. Evol. 36: 290-300and Altschul, S. F. et. al. (1990) J. Mol. Biol. 215:403. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (e.g. via their website).

Variant polypeptides therefore include naturally occurring allelicvariants. An allelic variant will generally be of human or non-humanmammal origin, such as bovine or porcine origin. Alternatively, avariant polypeptide can be a non-naturally occurring sequence. Anon-naturally occurring variant may thus be a modified version ofOSGPR114 or OSGPR78.

The amino acid sequence of OSGPR114 or OSGPR78 may be modified bydeletion and/or substitution and/or addition of single amino acids orgroups of amino acids as long as the modified polypeptide retains thecapability to function as a G-protein coupled receptor. Such amino acidchanges may occur in one, two or more of the intracellular domains ofOSGPR114 or OSGPR78 and/or one, two or more of the extracellular domainsof OSGPR114 or OSGPR78 and/or one, two or more of the transmembranedomains of OSGPR114 or OSGPR78.

Amino acid substitutions may thus be made, for example from 1, 2, 3, 4or 5 to 10, 20 or 30 substitutions. Conservative substitutions may bemade, for example according to the following Table. Amino acids in thesame block in the second column and preferably in the same line in thethird column may be substituted for each other.

ALIPHATIC Non-polar GAP ILV Polar-uncharged CSTM N Q Polar-charged D E KR AROMATIC HFWY

A variant polypeptide may be a shorter polypeptide. For example, apolypeptide of at least 20 amino acids or up to 50, 60, 70, 80, 100,150, 200, 250, or 300 amino acids in length may constitute a variantpolypeptide as long as it demonstrates the functionality of OSGPR114 orOSGPR78. A variant polypeptide may therefore lack one, two or moreintracellular domains and/or one, two or more extracellular domainsand/or one, two or more transmembrane domains. A variant polypeptide maythus be a fragment of the full-length polypeptide. A shortenedpolypeptide may comprise a ligand-binding region (N-terminalextracellular domain) and/or an effector binding region (C-terminalintracellular domain). Such fragments can be used to construct chimericreceptors, preferably with another 7-transmembrane G-protein coupledreceptor.

Variant polypeptides include polypeptides that are chemically modified,e.g. post-translationally modified. For example, such variantpolypeptides may be glycosylated or comprise modified amino acidresidues, e.g. phospho-amino acids. They may also be modified by theaddition of histidine residues, for example 6 or 8 His residues, or anepitope tag, for example a T7, HA, myc or flag tag, to assist theirpurification or detection. They may be modified by the addition of asignal sequence to promote insertion into the cell membrane.

The invention also utilizes nucleotide sequences that encode OSGPR114 orOSGPR78 or variants thereof as well as nucleotide sequences which arecomplementary thereto. The nucleotide sequence may be RNA or DNA,including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotidesequence is a DNA sequence, and most preferably, a cDNA sequence. Suchnucleotides can be isolated from human cells or synthesized according tomethods well known in the art, as described by way of example inSambrook et. al., Molecular Cloning: A Laboratory Manual, 2^(nd)edition, Cold Spring Harbour Laboratory Press, 1989. Typically a usefulpolynucleotide comprises a contiguous sequence of nucleotides which iscapable of hybridizing under selective conditions to the coding sequenceor the complement of the coding sequences of OSGPR114 or OSGPR78.

A polynucleotide can hydridize to the coding sequence or the complementof the coding sequences of OSGPR114 or OSGPR78 at a level significantlyabove background. Background hybridization may occur, for example,because of other cDNAs present in a cDNA library. The signal levelgenerated by the interaction between a polynucleotide and the codingsequence or complement of the coding sequence of OSGPR114 or OSGPR78 istypically at least 10 fold, preferably at least 100 fold, as intense asinteractions between other polynucleotides and the coding sequence ofOSGPR114 or OSGPR78. The intensity of interaction may be measured, forexample, by radiolabelling the probe, e.g. with ³²P Selectivehybridization may typically be achieved using conditions of lowstringency (0.3 M sodium chloride and 0.03 M sodium citrate at about 40°C., medium stringency (for example, 0.3 M sodium chloride and 0.03 Msodium citrate at about 50° C., or high stringency (for example, 0.03 Msodium chloride and 0.003 M sodium citrate at about 60° C.

The coding sequences of OSGPR114 or OSGPR78 may be modified by one ormore nucleotide substitutions, for example from 1, 2, 3, 4 or 5 to 10,25, 50 or 100 substitutions. The polynucleotides of OSGPR114 or OSGPR78may alternatively or additionally be modified by one or more insertionsand/or deletions and/or by an extension at either or both ends. Themodified polynucleotides generally encode polypeptides which haveG-protein coupled receptor activity or inhibit the activity of OSGPR114or OSGPR78. Degenerate substitutions may be made and/or substitutionsmay be made which would result in a conservative amino acid substitutionwhen the modified sequences are translated, for example as shown in theTable above.

A nucleotide sequence which is capable of selectively hybridizing to thecomplement of the DNA coding sequences of OSGPR114 or OSGPR78 willgenerally have at least 60%, at least 70%, at least 80%, at least 90%,at least 95%, at least 98% or at least 99% sequence identity to thecoding sequence of OSGPR114 or OSGPR78 over a region of at least 20,preferably at least 30, for instance at least 40, at least 60, morepreferably at least 100, 300, 600, 900 contiguous nucleotides, or mostpreferably over the full length. Methods of measuring nucleic acid andprotein homology are well known in the art. For example the UWGCGPackage provides the BESTFIT program which can be used to calculatehomology (Devereux J, et. al. (1984) Nucleic Acids Res 12:387-395).Similarly the PILEUP and BLAST algorithms can be used to line upsequences (for example, as described in Altschul, S. F. et. al. (1990)J. Mol. Biol. 215:403-410; Altschul, S. F., et. al. (1997) Nucleic AcidsRes. 25:3389-3402). Many different settings are possible for suchprograms. In accordance with the invention, the default settings may beused.

Any combination of the above mentioned degrees of sequence identity andminimum sizes may be used to define polynucleotides of the invention,with the more stringent combinations (i.e. higher sequence identity overlonger lengths) being preferred.

Thus, for example a polynucleotide which has at least 90% sequenceidentity over 25, preferably over 30 nucleotides forms one aspect of theinvention, as does a polynucleotide which has at least 95% sequenceidentity over 40 nucleotides.

Polynucleotides may be used as a primer, e.g. a PCR primer or a primerfor an alternative amplification reaction of a probe, e.g. labelled witha revealing label by conventional means for identifying mutations inOSGPR114 or OSGPR78 that may be implicated in diseases resulting fromabnormal control of cell proliferation. Fragments of polynucleotides maybe fused to the coding sequence of other proteins, preferably otherG-protein coupled receptors, to form a sequence coding for a fusionprotein.

Such primers, probes and other fragments will preferably be at least 10,preferably at least 15 or at least 20, for example at least 25, at least30 or at least 40 nucleotides in length. They will typically be up to40, 50, 60, 70, 100 or 150 nucleotides in length. Probes and fragmentscan be longer than 150 nucleotides in length, for example up to 200,300, 400, 500 nucleotides in length, or even up to a few nucleotides,such as five or ten nucleotides, short of the coding sequences ofOSGPR114 or OSGPR78.

The polynucleotides have utility in production of OSGPR114 or OSGPR78 orvariant polypeptides, which may take place in vitro, in vivo or ex vivo.The polynucleotides may be used as therapeutic agents in their ownright, in gene therapy techniques. The polynucleotides are cloned intoexpression vectors for these purposes. Such expression vectors areroutinely constructed in the art of molecular biology and may forexample involve the use of plasmid DNA and appropriate initiators,promoters, enhancers and other elements, such as for examplepolyadenylation signals which may be necessary, and which are positionedin the correct orientation, in order to allow for protein expression.Other suitable vectors would be apparent to a person skilled in the art.By way of further example in this regard, Molecular Cloning: aLaboratory Manual, 2001, 3^(rd) Edition, by Joseph Sambrook and PeterMacCallum (the former Maniatis Cloning manual) provides a good source.

Expression vectors comprise a polynucleotide encoding the desiredpolypeptide operably linked to a control sequence which is capable ofproviding for the expression of the coding sequence by a host cell. Theterm “operably linked” refers to a juxtaposition wherein the componentsdescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence, such as a promoter, “operablylinked” to a coding sequence is positioned in such a way that expressionof the coding sequence is achieved under conditions compatible with theregulatory sequence. Thus nucleic acid of this invention is operablylinked when it is placed into a functional relationship with anothernucleic acid sequence. For example, DNA for a presequence or secretoryleader is operably linked to DNA for a polypeptide if it is expressed asa preprotein which participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, operably linked means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading frame.

The vectors may be plasmid, virus or phage vectors provided with anorigin of replication, optionally a promoter for the expression of thesaid polynucleotide, and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid or aresistance gene for a fungal vector. Vectors may be used in vitro, forexample for the production of RNA or DNA or used to transfect ortransform a host cell, for example, a mammalian host cell. The vectorsmay also be adapted to be used in vivo, for example in a method of genetherapy.

This invention provides vectors comprising any nucleic acids encodingthe OSGPR114 or OSGPR78 receptors of this invention, including vectorsadapted for expression in a cell, which vector comprises the regulatoryelements necessary for expression of the nucleic acid in the celloperatively linked to the nucleic acid encoding the receptor so as topermit expression thereof. Furthermore this invention also providesvectors which are plasmids.

This invention further provides host cells comprising any of the vectorsdescribed herein. The host cell is typically a eukaryotic cell, amammalian cell, a human cell, an insect cell, a yeast cell or aprokaryotic cell, although is not limited to these. In one embodiment ofthis invention CHO, CHO-K1, RH7777, Jurkat, HCT4, or RBL243 cells areused. In another HeLa, ASPC-1, HEK-293, or COS7 cells are used.

Recombinant methods for synthesis of the OSGPR114 or OSGPR78 receptorsof this invention commence with the construction of a replicable vectorcontaining nucleic acid that encodes the OSGPR114 or OSGPR78 receptor.Vectors typically perform two functions in collaboration with compatiblehost cells. One function is to facilitate the cloning of the nucleicacid that encodes the OSGPR114 or OSGPR78 receptor, i.e., to produceusable quantities of the nucleic acid. The other function is to directthe expression of the OSGPR114 or OSGPR78 receptor. One or both of thesefunctions are performed by the vector-host system. The vectors willcontain different components depending upon the function they are toperform as well as the host cell that is selected.

This invention thus provides vectors that contain nucleic acid encodingthe OSGPR114 or OSGPR78 receptor. Typically, this will be DNA thatencodes the OSGPR114 or OSGPR78 receptor in its mature form linked atits amino terminus to a secretion signal. This secretion signalpreferably is the signal presequence that normally directs the insertionof the wild-type OSGPR114 or OSGPR78 receptor through the plasmamembrane. However, suitable secretion signals also include signals fromother receptors or from secreted polypeptides of the same or relatedspecies.

In instances where the expression of OSGPR114 or OSGPR78 would exert anundesired biological effect on the host cell if induced to accumulate inhigh concentration in the cell membrane during the growth phase, thispotential problem may be overcome by placing the nucleic acid encodingthe OSGPR114 or OSGPR78 receptor under the control of an induciblepromoter.

In the practice of this invention, for cloning vectors the OSGPR114 orOSGPR78 receptor-encoding nucleic acid ordinarily is present togetherwith a nucleic acid sequence that enables the vector to replicate in aselected host cell independent of the host chromosomes. This sequence isgenerally an origin of replication or an autonomously replicatingsequence. Such sequences are well-known for a variety of bacteria, yeastand higher eukaryotic cells. The origin from the well-known plasmidpBR322 is suitable for E. coli bacteria, the 2μ plasmid origin for yeastand various viral origins for mammalian cells (SV40, polyoma, adenovirusor bovine papilloma virus). Less desirably, DNA is cloned by insertioninto the genome of a host. This is readily accomplished with bacillusspecies, for example, by inserting into the vector DNA that iscomplementary to bacillus genomic DNA. Transfection of bacillus withthis vector results in homologous recombination with the genome andinsertion of the OSGPR114 or OSGPR78 receptor DNA. However, the recoveryof genomic DNA encoding the OSGPR114 or OSGPR78 receptor is more complexthan obtaining exogenously replicated viral or plasmid DNA becauserestriction enzyme digestion is required to recover the OSGPR114 orOSGPR78 receptor DNA from the genome of the cloning vehicle.

In the practice of this invention, expression and cloning vectors shouldcontain a selection gene, also termed a selectable marker. This is agene that encodes a protein necessary for the survival or growth of ahost cell transformed with the vector. The presence of this gene ensuresthe growth of only those host cells that express the inserts. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g. ampicillin, neomycin, blasticidin,G-418, mycophenolic acid, hygromycin B, bleomycin, phleomycin,methotrexate or tetracycline, (b) complement auxotrophic deficiences, or(c) supply critical nutrients not available from complex media, e.g. thegene encoding D-alanine racemase for bacilli.

A suitable selection gene for use in yeast is the TRP1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., 1979, “Nature”, 282: 39;Kingsman et al., 1979, “Gene”, 7: 141; or Tschemper et al., 1980,“Gene”, 10: 157). The TRP1 gene provides a selection marker for a mutantstrain of yeast lacking the ability to grow in the absence oftryptophan, for example ATCC No. 44076 or PEP41 (Jones, 1977,“Genetics”, 85: 12). The presence of the trp1 lesion in the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan. Similarly, leu2deficient yeast strains (ATCC 20,622 or 38,626) are complemented byknown plasmids bearing the LEU2 gene.

Examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR), thymidine kinase or proteins forneomycin resistance. Such markers enable the identification of cellsthat were competent to take up the OSGPR114 or OSGPR78 receptor nucleicacid. The mammalian cell transformants are placed under selectionpressure, which only the transformants are uniquely adapted to surviveby virtue of having taken up the marker. Selection pressure is imposedby culturing the transformants in successive rounds of cell culture, inwhich the concentration of selection agent in the medium is successivelyincreased, thereby leading to amplification of both the selection geneand the DNA encoding the OSGPR114 or OSGPR78 receptor. Increasedquantities of OSGPR114 or OSGPR78 receptor are synthesized from theamplified DNA.

For example, selection for DHFR transformed cells is conducted in aculture medium which lacks hypoxanthine, glycine, and thymidine. Anappropriate host cell in this case is the Chinese hamster ovary (CHO)cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub and Chasin, 1980, “Proc. Nat'l. Acad, Sci. USA” 77:4216.

A particularly useful DHFR is a mutant DHFR that is highly resistant tomethotrexate (MTX) (EP 117,060A). This selection agent can be used withany otherwise suitable host, notwithstanding the presence of endogenousDHFR. One simply includes sufficient MTX in the medium to inactivate allof the endogenous DHFR, whereupon MTX selection becomes solely afunction of amplification of the mutant DHFR DNA. Most eukaryotic cellswhich are capable of adsorbing MTX appear to be methotrexate sensitive.One such useful cell line is a CHO line, CHO-K1 (ATCC No. CCL 61).

Other methods, vectors and host cells suitable for adaptation to thesynthesis of the OSGPR114 or OSGPR78 receptor of this invention inrecombinant vertebrate cell culture are described in M. J. Gething etal., Nature 293: 620-625 (1981); N. Mantei et al., Nature 281: 40-46; EP117,060A; EP 117,058A; Molecular Cloning: a Laboratory Manual, 2001,3^(rd) Edition, by Joseph Sambrook and Peter MacCallum, (the formerManiatis Cloning manual) (e.g. ISBN 0-87969-577-3); and CurrentProtocols in Molecular Biology, Ed. Fred M. Ausubel, et. al. John Wiley& Sons (e.g. ISBN 0-471-50338-X).

Expression vectors of this invention, unlike cloning vectors, shouldcontain a promoter and/or other sequence that is recognized by the hostorganism for strong transcription of the OSGPR114 or OSGPR78 receptorencoding DNA. This is generally a promoter homologous to the intendedhost. In the case of vectors for higher eukaryotes, enhancer sequencesare useful for further increasing transcription from promoters. Unlikepromoters, enhancers do not need to be located 5′ to the OSGPR114 orOSGPR78 receptor encoding nucleic acid. Commonly used promoters forprokaryotes include the beta-lactamase and lactose promoter systems(Chang et al., 1978, “Nature”, 275: 615; and Goeddel et al., 1979,“Nature”, 281; 544), alkaline phosphatase, a tryptophan (trp) promotersystem (Goeddel 1980, “Nucleic Acids Res.” 8: 4057 and EPO Appln. Publ.No. 36,776) and hybrid promoters such as the tac promoter (H. de Boer etal., 1983, “Proc. Nat'l. Acad. Sci. USA” 80: 21-25). However, otherknown microbial promoters are suitable. Their nucleotide sequences havebeen published, thereby enabling a skilled worker operably to ligatethem to DNA encoding the OSGPR114 or OSGPR78 receptor in plasmid vectors(Siebenlist et al., 1980, “Cell” 20: 269) using linkers or adaptors tosupply any required restriction sites. Promoters for use in prokaryoticsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked to the DNA encoding the OSGPR114 or OSGPR78 receptor.

Suitable promoting sequences in yeast vectors for use in the practice ofthis invention include S. cerevisiae GAL4 and ADH promoters, and S.pombe nmt1 and adh promoters, and further include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., 1980, “J.Biol. Chem.”, 255: 2073) or other glycolytic enzymes (Hess et al., 1968,“J. Adv. Enzyme Reg.”, 7: 149; and Holland, 1978, “Biochemistry”, 17:4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters for use in the practice of this invention, whichhave the additional advantage of transcription controlled by growthconditions, are the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and the aforementioned metallothionein andglyceralidehyde-3-phosphate dehydrogenase, as well as enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in R.Hitzeman et al., EP 73,657A.

In the practice of this invention, transcription from vectors inmammalian host cells is controlled by promoters and/or enhancersobtained from the genomes of bovine papilloma virus, vaccinia virus,polyoma virus, adenovirus 2, retroviruses, hepatitus-B virus,cytomegalovirus (e.g. IE promoter), spleen focus forming virus, murinestem cell virus, Moloney murine leukemia virus (e.g. MMLV LTR), SimianVirus 40 (SV40), HSV (such as the HSV IE promoters), or HPV(particularly the HPV upstream regulatory region (URR)), operably linkedto the OSGPR114 or OSGPR78 receptor nucleic acid. The early and latepromoters of the SV40 virus are as conveniently obtained as an SV40restriction fragment, which also contains the SV40 viral origin ofreplication (Fiers et al., 1978, “Nature”, 273: 113). Mammalianpromoters also include the metallothionein promoter, which can beinduced in response to heavy metals such as cadmium. Of course,promoters or enhancers from the host cell or related species also areuseful herein. Mammalian promoters, such as P-actin promoters, may beused. Tissue specific promoters, for example adipose or pancreatic cellspecific promoters, may also be used. A suitable mammalian expressionvector for practice of this invention is pcDNA3.1. Retrovirus vectorsmay also be used in the practice of this invention (e.g. rous sarcomavirus (RSV) LTR promoter), including those with inducible elements, e.g.tetracycline responsive elements.

The vector may further include sequences flanking the polynucleotidewhich comprise sequences homologous to eukaryotic genomic sequences,preferably mammalian genomic sequences, or viral genomic sequences. Thiswill allow the introduction of the relevant polynucleotides into thegenome of eukaryotic cells or viruses by homologous recombination. Inparticular, a plasmid vector comprising the expression cassette flankedby viral sequences can be used to prepare a viral vector suitable fordelivering the polynucleotides of the invention to a mammalian cell.Retrovirus vectors for example may be used to stably integrate thepolynucleotide into the host genome. Replication-defective adenovirusvectors by contrast remain episomal and therefore allow transientexpression.

Expression vectors used in eukaryotic host cells of this invention(yeast, fungi, insect, plant, animal or human) will also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the3′-untranslated regions of eukaryotic or viral cDNAs. These regionscontain regions that are transcribed as polyadenylated segments in theuntranslated portion of the mRNA encoding the OSGPR114 or OSGPR78receptor. The 3′ untranslated regions also include transcriptiontermination sites.

Cells are transformed or transfected with the vectors to express theOSGPR114 or OSGPR78 polypeptide or variants thereof. Such cells may beeukaryotic or prokaryotic. They include transient or, preferably, stablehigher eukaryotic cell lines such as mammalian cells or insect cells,lower eukaryotic cells such as yeast, and prokaryotic cells such asbacterial cells. Particular examples of cells which may be used toexpress OSGPR114 or OSGPR78 or a variant polypeptide include mammaliancells such as HEK293T, CHO, CHO-K1, HeLa, ASPC-1, RH7777, Jurkat, HCT4,RBL243 and COS7 cells. Preferably the cell line selected will be onewhich is not only stable, but also allows for mature glycosylation andcell surface expression of OSGPR114 or OSGPR78 polypeptide or a variant.Cells such as adipose or pancreatic cells expressing OSGPR114 or OSGPR78receptors or a variant polypeptide may be used in screening assays.Expression may be achieved in transformed oocytes. The OSGPR114 orOSGPR78 polypeptides or a variant may be expressed in cells such asthose of adipose or pancreatic tissue of a transgenic non-human animal,preferably a rodent such as a mouse.

Suitable host cells for cloning or expressing the vectors herein areprokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gramnegative or gram positive organisms, for example E. coli or bacilli. Apreferred cloning host is E. coli 294 (ATCC 31,446) although other gramnegative or gram positive prokaryotes such as E. coli B, E. coli X1776(ATCC 31,537), E. coli W3110 (ATCC 27,325), Pseudomonas species, orSerratia marcesans are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for the OSGPR114 or OSGPR78 receptorencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species and strains are commonlyavailable and useful herein.

The preferred host cells for the expression of functional OSGPR114 orOSGPR78 receptors of this invention are cultures of cells derived frommulticellular organisms. OSGPR114 or OSGPR78 receptors or variantsthereof may contain hydrophobic regions that are incompatible with lowermicroorganisms, require complex processing to properly form disulfidebonds or require subunit processing. In addition, it may be desirable toglycosylate the receptor in a fashion similar to the native receptor.All of these functions can be best performed by higher eukaryotic cells.In principle, any higher eukaryotic cell culture is workable, whetherfrom vertebrate or invertebrate culture, although cells from mammalssuch as humans are preferred. Propagation of such cells in culture isper se well known. See Tissue Culture, Academic Press, Kruse andPatterson, editors (1973), or Culture of Animal Cells: A Manual of BasicTechnique, 4th Ed. Author, R. Ian Freshney, John Wiley & Sons., (2000).Examples of useful mammalian host cell lines are VERO and HeLa cells,human 239 cells, quail QT6 cells, NIH-3T3 cells, Chinese hamster ovarycell lines (CHO), W138, BHK, COS-7, HEK293, HeLa, ASPC-1, RH7777,Jurkat, HCT4, RBL243, and MDCK cell lines.

Thus, this invention also provides a cell comprising an OSGPR114 orOSGPR78 receptor. The cell of this invention can be eukaryotic,mammalian, human, insect or yeast. The cell comprising the OSGPR114 orOSGPR78 receptor of this invention can be a stable or transienttransfectant.

In embodiments of this invention where purification of the OSGPR114 orOSGPR78 receptor is required, for example from a detergent solubilizedmembrane preparation containing the OSGPR114 or OSGPR78 receptor, theOSGPR114 or OSGPR78 receptor is readily purified by any of the proteinpurification techniques commonly practiced in the art, e.g.immunoaffinity chromatography. The recombinant OSGPR114 or OSGPR78receptor can also be engineered to contain a structural element orepitope to assist in its purification, e.g. poly-histidine,calmodulin-binding peptide, glutathione-S-transferase, ormaltose-binding protein.

The present invention is concerned in particular with the use ofOSGPR114 or OSGPR78 or a functional variant in screening methods toidentify agents that may act as modulators of OSGPR114 or OSGPR78receptor activity and, in particular, agents that may act as modulatorsof cell proliferation, particularly inhibitors of cell proliferationthat can inhibit tumor growth. Modulators identified by such screeningmethods are useful in the treatment OSGPR114 or OSGPR78-mediateddiseases, including diseases or conditions such as cancer, obesity,certain diseases of the cardiovascular and respiratory systems,including artherosclerosis, restenosis and acetylcholine induced airwayhyperresponsiveness, wound healing, reproductive function, abnormalimmune system regulation, abnormalities in neuronal growth, survival andsignaling, and renal failure associated with renal ischemia. By the termOSGPR114 or OSGPR78-mediated disease, it is meant those diseases orconditions where the modulation of OSGPR114 or OSGPR78 by agonists orantagonists results in a beneficial modification of the disease state orcondition.

Any suitable form of assay may be employed to identify a modulator ofOSGPR114 or OSGPR78 activity, and/or of cell proliferation or tumorgrowth. In general terms, such screening methods involve contactingOSGPR114 or OSGPR78 or a variant polypeptide with a test compound andthen determining receptor activity. G-protein activation, for exampleGi/o-protein or Gs-protein activation, may be determined therefore.Where a test compound affects receptor activity, its effect on cellproliferation can be determined by contacting cells with the testcompound and measuring effects on cell proliferation by any of themethods well known in the art (e.g. see Experimental Details below).

OSGPR114 or OSGPR78 modulator activity can be determined in vitro or invivo by contacting cells expressing OSGPR114 or OSGPR78 or a variantpolypeptide with an agent under test and by monitoring the effectmediated by OSGPR114 or OSGPR78 or variant polypeptide. Thus, a testagent may be contacted with isolated cells which express OSGPR114 orOSGPR78 or a variant polypeptide. The cells may be provided in culture.Alternatively, cells may be disrupted and cell membranes isolated andused. OSGPR114 or OSGPR78 receptor protein may also be solubilized withthe use of detergents (e.g. non-ionic detergents, such as digitonin),and the receptor purified and reconstituted in lipid vesicles, withother purified proteins as required. Such reconstitutedligand-stimulated GPCRs, that can activate second messenger systems, arewell known in the art, and can be used in the practice of thisinvention.

The OSGPR114 or OSGPR78 or variant polypeptide may be naturally orrecombinantly expressed. Preferably, an assay is carried out in vitrousing cells expressing recombinant polypeptide or using membranes fromsuch cells. Suitable eucaryotic and procaryotic cells are discussedabove. In one embodiment, a cell type known to naturally expressOSGPR114 or OSGPR78 receptors is used, e.g. an ovarian, liver, colon,lung, adipose or breast cell.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a mammalian OSGPR114 or OSGPR78 receptorwhich comprises contacting cells containing DNA encoding, and expressingon their cell surface, the mammalian OSGPR114 or OSGPR78 receptor,wherein such cells do not normally express the mammalian OSGPR114 orOSGPR78 receptor, with the compound under conditions suitable forbinding, and detecting specific binding of the chemical compound to themammalian OSGPR114 or OSGPR78 receptor.

This invention further provides a process for identifying a chemicalcompound which specifically binds to a mammalian OSGPR114 or OSGPR78receptor which comprises contacting a membrane preparation from cellscontaining DNA encoding, and expressing on their cell surface, themammalian OSGPR114 or OSGPR78 receptor, wherein such cells do notnormally express the mammalian OSGPR114 or OSGPR78 receptor, with thecompound under conditions suitable for binding, and detecting specificbinding of the chemical compound to the mammalian OSGPR114 or OSGPR78receptor.

In a further embodiment of these processes for identifying a chemicalcompound which specifically binds to a mammalian OSGPR114 or OSGPR78receptor the contacting of the cells or membrane preparation with thecompound is performed in the presence of a ligand for the OSGPR114 orOSGPR78 receptor, wherein the ligand is an LPA compound. In oneembodiment, the LPA is selected from myristoyl lysophosphatidic acid,oleoyl lysophosphatidic acid, palmitoyl lysophosphatidic acid, andstearoyl lysophosphatidic acid, or a functional analog or homolog of oneof these compounds.

In one embodiment, the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In another embodiment, the mammalianOSGPR114 or OSGPR78 receptor has the same or substantially the sameamino acid sequence as the human OSGPR114 or OSGPR78 receptor of FIG. 2.

In another embodiment, the mammalian OSGPR114 or OSGPR78 receptor is amouse OSGPR114 or OSGPR78 receptor. In another embodiment, the mammalianOSGPR114 or OSGPR78 receptor has the same or substantially the sameamino acid sequence as the mouse OSGPR114 or OSGPR78 receptor withGenBank Accession numbers AY255621 and NM_(—)175116.

In another embodiment, the mammalian OSGPR114 or OSGPR78 receptor is arat, dog, bovine, porcine or monkey OSGPR114 or OSGPR78 receptor.

In one embodiment, the compound is not previously known to bind to amammalian OSGPR114 or OSGPR78 receptor. In one embodiment, the cell isan insect cell. In one embodiment, the cell is a mammalian cell. Inanother embodiment, the cell is a COS-7, ASPC-1, RH7777, Jurkat, HCT4,or RBL243 cell, a 293 human embryonic kidney cell, a CHO cell, a NIH-3T3cell, a mouse Y1 cell, or a LM(tk−) cell. In another embodiment, thecompound is a compound not previously known to bind to a mammalianOSGPR114 or OSGPR78 receptor. This invention provides a compoundidentified by the preceding process of this invention.

This invention provides an assay process for identifying a compound thatspecifically binds to a mammalian OSGPR114 or OSGPR78 receptor, saidprocess comprising: providing either (a) two samples of cells expressingon their cell surface the mammalian OSGPR114 or OSGPR78 receptor, or (b)two samples of a membrane preparation from said cells; contacting onesample with a lysophosphatidic acid ligand known to bind to thereceptor, under conditions suitable for binding of said ligand to thereceptor, in the presence of a test compound; contacting the secondsample with said ligand, under conditions suitable for binding of saidligand to the receptor, in the absence of the test compound; measuringthe specific binding of the ligand to the receptor in the presence ofthe compound; measuring the specific binding of the ligand to thereceptor in the absence of the compound; and comparing the binding inthe presence and in the absence of the compound being tested, wherein adifference in comparison indicates that the compound binds to themammalian OSGPR114 or OSGPR78 receptor. The compound identified may be,for example, a competitive or non-competitive antagonist, or a partialagonist.

This invention still further provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa mammalian OSGPR114 or OSGPR78 receptor which comprises separatelycontacting cells expressing on their cell surface the mammalian OSGPR114or OSGPR78 receptor, wherein such cells do not normally express themammalian OSGPR114 or OSGPR78 receptor (i.e. the host cells transformedor transfected with vectors to express the OSGPR114 or OSGPR78polypeptide or variants thereof do not express these polypeptides atsignificant levels prior to introduction of the vector), with both thechemical compound and an LPA ligand, known to bind to the receptor, andwith only the ligand, under conditions suitable for binding of such aligand to the receptor, and detecting specific binding of the chemicalcompound to the mammalian OSGPR114 or OSGPR78 receptor, a decrease inthe binding of the ligand to the mammalian OSGPR114 or OSGPR78 receptorin the presence of the chemical compound being tested indicating thatsuch chemical compound binds to the mammalian OSGPR114 or OSGPR78receptor.

This invention also provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianOSGPR114 or OSGPR78 receptor which comprises separately contacting amembrane preparation from cells expressing on their cell surface themammalian OSGPR114 or OSGPR78 receptor, wherein such cells do notnormally express the mammalian OSGPR114 or OSGPR78 receptor, with boththe chemical compound and an LPA ligand, known to bind to the receptor,and with only the ligand, under conditions suitable for binding of sucha ligand to the receptor, and detecting specific binding of the chemicalcompound to the mammalian OSGPR114 or OSGPR78 receptor, a decrease inthe binding of the ligand to the mammalian OSGPR114 or OSGPR78 receptorin the presence of the chemical compound being tested indicating thatsuch chemical compound binds to the mammalian OSGPR114 or OSGPR78receptor.

In specific embodiments of these processes the LPA ligand is a naturallyoccurring LPA. In a further embodiment, LPA is selected from myristoyllysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid, or afunctional analog or homolog of one of these compounds.

In one embodiment, the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In another embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a rat OSGPR114 or OSGPR78 receptor. Inanother embodiment, the mammalian OSGPR114 or OSGPR78 receptor is amouse OSGPR114 or OSGPR78 receptor. In a further embodiment, the cell isan insect cell. In another embodiment, the cell is a mammalian cell. Inanother embodiment, the cell is a yeast cell. In another embodiment, thecell is a ASPC-1, RH7777, Jurkat, HCT4, RBL243 or COS-7 cell, 293 humanembryonic kidney cell (HEK-293), a CHO cell, a NIH-3T3 cell, a mouse Y1cell, or a LM(tk−) cell. In another embodiment, the compound is notpreviously known to bind to a mammalian OSGPR114 or OSGPR78 receptor.This invention provides a compound identified by the preceding processesof this invention.

This invention provides a method of screening a plurality of compoundsnot known to bind to a mammalian OSGPR114 or OSGPR78 receptor toidentify a compound which specifically binds to the mammalian OSGPR114or OSGPR78 receptor, said process comprising: providing either (a) twosamples of cells expressing on their cell surface the mammalian OSGPR114or OSGPR78 receptor, or (b) two samples of a membrane preparation fromsaid cells; contacting one sample with a lysophosphatidic acid ligandknown to bind to the receptor, under conditions suitable for binding ofsaid ligand to the receptor, in the presence of the plurality ofcompounds not known to bind to the receptor; contacting the secondsample with said ligand, under conditions suitable for binding of saidligand to the receptor, in the absence of the plurality of compounds;measuring specific binding of the ligand to the receptor in the presenceof the plurality of compounds; measuring specific binding of the ligandto the receptor in the absence of the plurality of compounds; comparingthe binding in the presence and in the absence of the plurality ofcompounds, wherein a difference in the compared binding resultsindicates that one or more compounds in the plurality of compounds bindsto the mammalian OSGPR114 or OSGPR78 receptor; and determining, when adifference in the compared binding is found, the binding to themammalian OSGPR114 or OSGPR78 receptor of each compound included in theplurality of compounds, to identify any compound included therein whichspecifically binds to the mammalian OSGPR114 or OSGPR78 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian OSGPR114 or OSGPR78 receptorto identify a compound which specifically binds to the mammalianOSGPR114 or OSGPR78 receptor, which comprises (a) contacting cellstransfected with, and expressing, DNA encoding the mammalian OSGPR114 orOSGPR78 receptor with a compound known to bind specifically to themammalian OSGPR114 or OSGPR78 receptor (e.g. a ligand); (b) contactingthe cells of step (a) with the plurality of compounds not known to bindspecifically to the mammalian OSGPR114 or OSGPR78 receptor, underconditions permitting binding of compounds known to bind to themammalian OSGPR114 or OSGPR78 receptor; (c) determining whether thebinding of the compound known to bind to the mammalian OSGPR114 orOSGPR78 receptor is reduced in the presence of the plurality ofcompounds, relative to the binding of the compound in the absence of theplurality of compounds; and if so (d) separately determining the bindingto the mammalian OSGPR114 or OSGPR78 receptor of each compound includedin the plurality of compounds, so as to thereby identify any compoundincluded therein which specifically binds to the mammalian OSGPR114 orOSGPR78 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian OSGPR114 or OSGPR78 receptorto identify a compound which specifically binds to the mammalianOSGPR114 or OSGPR78 receptor, which comprises (a) contacting a membranepreparation from cells transfected with, and expressing, DNA encodingthe mammalian OSGPR114 or OSGPR78 receptor with a compound known to bindspecifically to the mammalian OSGPR114 or OSGPR78 receptor (e.g. aligand), and with the plurality of compounds not known to bindspecifically to the mammalian OSGPR114 or OSGPR78 receptor, underconditions permitting binding of compounds known to bind to themammalian OSGPR114 or OSGPR78 receptor; (b) determining whether thebinding of a compound known to bind to the mammalian OSGPR114 or OSGPR78receptor is reduced in the presence of the plurality of compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (c) separately determining the binding to themammalian OSGPR114 or OSGPR78 receptor of each compound included in theplurality of compounds, so as to thereby identify any compound includedtherein which specifically binds to the mammalian OSGPR114 or OSGPR78receptor.

In specific embodiments of these methods the compound known to bind tothe mammalian OSGPR114 or OSGPR78 receptor is an LPA ligand. In oneembodiment it is a naturally occurring LPA. In a further embodiment, LPAis selected from myristoyl lysophosphatidic acid, oleoyllysophosphatidic acid, palmitoyl lysophosphatidic acid, and stearoyllysophosphatidic acid, or a functional analog or homolog of one of thesecompounds.

In one embodiment, the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In a further embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a rat OSGPR114 or OSGPR78 receptor. Inanother embodiment, the mammalian OSGPR114 or OSGPR78 receptor is amouse OSGPR114 or OSGPR78 receptor. In another embodiment, the cell is amammalian cell. In another embodiment, the cell is a yeast cell. Inanother embodiment the cell is a CHO, RH7777, Jurkat, HCT4, RBL243, orCOS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell, a CHOcell, a mouse Y1 cell, or an NIH-3T3 cell.

This invention provides a method of detecting expression of a mammalianOSGPR114 or OSGPR78 receptor by detecting the presence of mRNA codingfor the mammalian OSGPR114 or OSGPR78 receptor which comprises obtainingtotal mRNA from the cell and contacting the mRNA so obtained with anucleic acid probe according to this invention under hybridizingconditions, detecting the presence of mRNA hybridized to the probe, andthereby detecting the expression of the mammalian OSGPR114 or OSGPR78receptor by the cell.

This invention provides a method of detecting the presence of amammalian OSGPR114 or OSGPR78 receptor on the surface of a cell whichcomprises contacting the cell with an antibody according to thisinvention under conditions permitting binding of the antibody to thereceptor, detecting the presence of the antibody bound to the cell, andthereby detecting the presence of the mammalian OSGPR114 or OSGPR78receptor on the surface of the cell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of mammalian OSGPR114 or OSGPR78receptors which comprises producing a transgenic, nonhuman mammal inaccordance with this invention whose levels of mammalian OSGPR114 orOSGPR78 receptor activity are varied by use of an inducible promoterwhich regulates mammalian OSGPR114 or OSGPR78 receptor expression.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of mammalian OSGPR114 or OSGPR78receptors which comprises producing a panel of transgenic, nonhumanmammals in accordance with this invention each expressing a differentamount of mammalian OSGPR114 or OSGPR78 receptor.

This invention provides a method for identifying an antagonist capableof alleviating an abnormality wherein the abnormality is alleviated bydecreasing the activity of a mammalian OSGPR114 or OSGPR78 receptorcomprising administering a compound to a transgenic, nonhuman mammalaccording to this invention, and determining whether the compoundalleviates any physiological and/or behavioral abnormality displayed bythe transgenic, nonhuman mammal as a result of overactivity of amammalian OSGPR114 or OSGPR78 receptor, the alleviation of suchabnormality identifying the compound as an antagonist. In oneembodiment, the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In a further embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a rat OSGPR114 or OSGPR78 receptor. Inanother embodiment, the mammalian OSGPR114 or OSGPR78 receptor is amouse OSGPR114 or OSGPR78 receptor. The invention provides an antagonistidentified by the preceding method according to this invention. Thisinvention provides a composition, e.g. a pharmaceutical composition,comprising an antagonist according to this invention and a carrier, e.g.a pharmaceutically acceptable carrier. This invention provides a methodof treating an abnormality in a subject wherein the abnormality isalleviated by decreasing the activity of a mammalian OSGPR114 or OSGPR78receptor which comprises administering to the subject an effectiveamount of the pharmaceutical composition according to this invention soas to thereby treat the abnormality. In one embodiment of this inventionthe abnormality is a tumor.

This invention provides a method for identifying an agonist capable ofalleviating an abnormality in a subject wherein the abnormality isalleviated by increasing the activity of a mammalian OSGPR114 or OSGPR78receptor comprising administering a compound to a transgenic, nonhumanmammal according to this invention, and determining whether the compoundalleviates any physiological and/or behavioral abnormality displayed bythe transgenic, nonhuman mammal, the alleviation of such an abnormalityidentifying the compound as an agonist. In one embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a human OSGPR114 or OSGPR78 receptor. Ina further embodiment, the mammalian OSGPR114 or OSGPR78 receptor is arat OSGPR114 or OSGPR78 receptor. In another embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a mouse OSGPR114 or OSGPR78 receptor.This invention provides an agonist identified by the preceding methodaccording to this invention. This invention provides a composition, e.g.a pharmaceutical composition, comprising an agonist identified by themethod according to this invention and a carrier, e.g. apharmaceutically acceptable carrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by increasing the activity of amammalian OSGPR114 or OSGPR78 receptor which comprises administering tothe subject an effective amount of the pharmaceutical compositionaccording to this invention so as to thereby treat the abnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific mammalian allele ofOSGPR114 or OSGPR78 which comprises: (a) obtaining DNA of subjectssuffering from the disorder; (b) performing a restriction digest of theDNA with a panel of restriction enzymes; (c) electrophoreticallyseparating the resulting DNA fragments on a sizing gel; (d) contactingthe resulting gel with a nucleic acid probe capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding a mammalian OSGPR114 or OSGPR78 receptorand labeled with a detectable marker; (e) detecting labeled bands whichhave hybridized to the DNA encoding a mammalian OSGPR114 or OSGPR78receptor to create a unique band pattern specific to the DNA of subjectssuffering from the disorder; (f) repeating steps (a)-(e) with DNAobtained for diagnosis from subjects not yet suffering from thedisorder; and (g) comparing the unique band pattern specific to the DNAof subjects suffering from the disorder from step (e) with the bandpattern from step (f) for subjects not yet suffering from the disorderso as to determine whether the patterns are the same or different andthereby diagnose predisposition to the disorder if the patterns are thesame.

In one embodiment, the disorder is a disorder associated with theactivity of a specific mammalian OSGPR114 or OSGPR78 receptor allele isdiagnosed.

This invention provides a method of preparing a purified mammalianOSGPR114 or OSGPR78 receptor according to this invention whichcomprises: (a) culturing cells which express the mammalian OSGPR114 orOSGPR78 receptor; (b) recovering the mammalian OSGPR114 or OSGPR78receptor from the cells; and (c) purifying the mammalian OSGPR114 orOSGPR78 receptor so recovered.

This invention provides a method of preparing the purified mammalianOSGPR114 or OSGPR78 receptor according to this invention whichcomprises: (a) inserting a nucleic acid encoding the mammalian OSGPR114or OSGPR78 receptor into a suitable expression vector; (b) introducingthe resulting vector into a suitable host cell; (c) placing theresulting host cell in suitable conditions permitting the production ofthe mammalian OSGPR114 or OSGPR78 receptor; (d) recovering the mammalianOSGPR114 or OSGPR78 receptor so produced; and optionally (e) isolatingand/or purifying the mammalian OSGPR114 or OSGPR78 receptor sorecovered.

This invention provides a process for determining whether a chemicalcompound is a mammalian OSGPR114 or OSGPR78 receptor agonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian OSGPR114 or OSGPR78 receptor with the compound underconditions permitting the activation of the mammalian OSGPR114 orOSGPR78 receptor, and detecting any increase in mammalian OSGPR114 orOSGPR78 receptor activity, so as to thereby determine whether thecompound is a mammalian OSGPR114 or OSGPR78 receptor agonist.

This invention provides a process for determining whether a chemicalcompound is a mammalian OSGPR114 or OSGPR78 receptor antagonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian OSGPR114 or OSGPR78 receptor with the compound in thepresence of a known mammalian OSGPR114 or OSGPR78 receptor agonist,under conditions permitting the activation of the mammalian OSGPR114 orOSGPR78 receptor, and detecting any decrease in mammalian OSGPR114 orOSGPR78 receptor activity, so as to thereby determine whether thecompound is a mammalian OSGPR114 or OSGPR78 receptor antagonist.

In one embodiment, the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In another embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a rat OSGPR114 or OSGPR78 receptor. Inanother embodiment, the mammalian OSGPR114 or OSGPR78 receptor is amouse OSGPR114 or OSGPR78 receptor.

This invention provides a composition, for example a pharmaceuticalcomposition, which comprises an amount of a mammalian OSGPR114 orOSGPR78 receptor agonist determined by a process according to thisinvention effective to increase activity of a mammalian OSGPR114 orOSGPR78 receptor and a carrier, for example, a pharmaceuticallyacceptable carrier. In one embodiment, the mammalian OSGPR114 or OSGPR78receptor agonist is not previously known.

This invention provides a composition, for example a pharmaceuticalcomposition, which comprises an amount of a mammalian OSGPR114 orOSGPR78 receptor antagonist determined by a process according to thisinvention effective to reduce activity of a mammalian OSGPR114 orOSGPR78 receptor and a carrier, for example, a pharmaceuticallyacceptable carrier. In one embodiment, the mammalian OSGPR114 or OSGPR78receptor antagonist is not previously known.

This invention provides a method of preparing a composition comprising acompound which specifically binds to a mammalian OSGPR114 or OSGPR78receptor, which comprises identifying a compound that specifically bindsto a mammalian OSGPR114 or OSGPR78 receptor by a process comprising:providing either (a) two samples of cells expressing on their cellsurface the mammalian OSGPR114 or OSGPR78 receptor, or (b) two samplesof a membrane preparation from said cells; contacting one sample with alysophosphatidic acid ligand known to bind to the receptor, underconditions suitable for binding of said ligand to the receptor, in thepresence of a test compound; contacting the second sample with saidligand, under conditions suitable for binding of said ligand to thereceptor, in the absence of the test compound; measuring specificbinding of the ligand to the receptor in the presence of the compound;measuring specific binding of the ligand to the receptor in the absenceof the compound; and comparing the binding in the presence and in theabsence of the compound being tested, wherein a difference in thebinding of the ligand to the mammalian OSGPR114 or OSGPR78 receptorindicates that the compound binds to the mammalian OSGPR114 or OSGPR78receptor; and admixing the compound so identified, or a functionalanalog or homolog of said compound, with a carrier, thereby preparingsaid composition.

This invention provides a method of preparing a composition, for examplea pharmaceutical composition, comprising a chemical compound whichspecifically binds to a mammalian OSGPR114 or OSGPR78 receptor, whichcomprises separately contacting cells expressing on their cell surfacethe mammalian OSGPR114 or OSGPR78 receptor, wherein such cells do notnormally express the mammalian OSGPR114 or OSGPR78 receptor, with both atest chemical compound and an LPA ligand, known to bind to the receptor,and with only the ligand, under conditions suitable for binding of sucha ligand to the receptor, and detecting specific binding of the testchemical compound to the mammalian OSGPR114 or OSGPR78 receptor, adecrease in the binding of the ligand to the mammalian OSGPR114 orOSGPR78 receptor in the presence of the test chemical compoundindicating that said test chemical compound binds specifically to themammalian OSGPR114 or OSGPR78 receptor, and admixing the test chemicalso identified, or a functional analog or homolog of said test chemical,with a carrier, thereby preparing said composition. In one embodiment ofthis method the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In another embodiment it is a rat or mouseOSGPR114 or OSGPR78 receptor.

This invention provides a method of preparing a composition, for examplea pharmaceutical composition, comprising a chemical compound whichspecifically binds to a mammalian OSGPR114 or OSGPR78 receptor, whichcomprises separately contacting a membrane preparation from cellsexpressing on their cell surface the mammalian OSGPR114 or OSGPR78receptor, wherein such cells do not normally express the mammalianOSGPR114 or OSGPR78 receptor, with both a test chemical compound and anLPA ligand, known to bind to the receptor, and with only the ligand,under conditions suitable for binding of such a ligand to the receptor,and detecting specific binding of the test chemical compound to themammalian OSGPR114 or OSGPR78 receptor, a decrease in the binding of theligand to the mammalian OSGPR114 or OSGPR78 receptor in the presence ofthe test chemical compound indicating that said test chemical compoundbinds specifically to the mammalian OSGPR114 or OSGPR78 receptor, andadmixing the test chemical so identified, or a functional analog orhomolog of said test chemical, with a carrier, thereby preparing saidcomposition. In one embodiment of this method the mammalian OSGPR114 orOSGPR78 receptor is a human OSGPR114 or OSGPR78 receptor. In anotherembodiment it is a rat or mouse OSGPR114 or OSGPR78 receptor.

This invention provides a process for determining whether a chemicalcompound specifically binds to and modulates activation of a mammalianOSGPR114 or OSGPR78 receptor, said process comprising: providing twosamples of cells expressing on their cell surface the mammalian OSGPR114or OSGPR78 receptor, wherein activation of the receptor produces asecond messenger response; contacting one sample, in the presence of atest compound, with a second compound known to activate the receptor,under conditions suitable for activation of the receptor; contacting thesecond sample, in the absence of the test compound, with the secondcompound known to activate the receptor, under conditions suitable foractivation of the receptor; measuring the second messenger response inthe presence of the test compound, measuring the second messengerresponse in the absence of the test compound; and comparing the secondmessenger response in the presence and in the absence of the compoundbeing tested, wherein a difference in the second messenger response fromthe mammalian OSGPR114 or OSGPR78 receptor indicates that the compoundmodulates activation of a mammalian OSGPR114 or OSGPR78 receptor. Thecompound identified may, for example, antagonize activation of thereceptor by the second compound, either competitively ornon-competitively, or may be an agonist or a partial agonist.

This invention provides a process for determining whether a chemicalcompound specifically binds to and activates a mammalian OSGPR114 orOSGPR78 receptor, which comprises contacting cells producing a secondmessenger response and expressing on their cell surface the mammalianOSGPR114 or OSGPR78 receptor, wherein such cells do not normally expressthe mammalian OSGPR114 or OSGPR78 receptor, with the chemical compoundunder conditions suitable for activation of the mammalian OSGPR114 orOSGPR78 receptor, and measuring the second messenger response in thepresence and in the absence of the chemical compound, a change, e.g. anincrease, in the second messenger response in the presence of thechemical compound indicating that the compound activates the mammalianOSGPR114 or OSGPR78 receptor.

In one embodiment, the second messenger response comprises chloridechannel activation and the change in second messenger is an increase inthe level of chloride current. In another embodiment, the secondmessenger response comprises a change in intracellular calcium levelsand the change in second messenger is an increase in the measure ofintracellular calcium. In another embodiment, the second messengerresponse comprises release of inositol phosphate and the change insecond messenger is an increase in the level of inositol phosphate. Inanother embodiment, the second messenger response comprises release ofarachidonic acid and the change in second messenger is an increase inthe level of arachidonic acid. In yet another embodiment, the secondmessenger response comprises GTPγS ligand binding and the change insecond messenger is an increase in GTPγS ligand binding. In anotherembodiment, the second messenger response comprises activation of MAPkinase and the change in second messenger response is an increase in MAPkinase activation. In a further embodiment, the second messengerresponse comprises cAMP accumulation and the change in second messengerresponse is an increase in cAMP accumulation. In another embodiment, thesecond messenger response comprises a change in intracellular sodium ionlevels and the change in second messenger is an increase in the measureof intracellular sodium ions. In another embodiment, the secondmessenger response comprises a change in intracellular potassium ionlevels and the change in second messenger is an increase in the measureof intracellular potassium ions.

Where the second messenger response being measured comprises GTPγSligand binding, the key GPCR activation step of guanine nucleotideexchange on the G-protein α-subunit is being assessed. This is a veryearly event in the signal transduction cascade, and as such is anattractive event to monitor, because it is less subject to amplificationor modification than more distal events that can be used to monitorOSGPR114 or OSGPR78 receptor activity.

This invention provides a process for determining whether a chemicalcompound specifically binds to and inhibits activation of a mammalianOSGPR114 or OSGPR78 receptor, which comprises separately contactingcells producing a second messenger response and expressing on their cellsurface the mammalian OSGPR114 or OSGPR78 receptor, wherein such cellsdo not normally express the mammalian OSGPR114 or OSGPR78 receptor, withboth the chemical compound and a second chemical compound known toactivate the mammalian OSGPR114 or OSGPR78 receptor, and with only thesecond chemical compound, under conditions suitable for activation ofthe mammalian OSGPR114 or OSGPR78 receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a smaller change, e.g. increase, in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian OSGPR114 or OSGPR78 receptor.

In one embodiment, the second messenger response comprises chloridechannel activation and the change in second messenger response is asmaller increase in the level of chloride current in the presence ofboth the chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another embodiment,the second messenger response comprises change in intracellular calciumlevels and the change in second messenger response is a smaller increasein the measure of intracellular calcium in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound. In another embodiment, the secondmessenger response comprises release of inositol phosphate and thechange in second messenger response is a smaller increase in the levelof inositol phosphate in the presence of both the chemical compound andthe second chemical compound than in the presence of only the secondchemical compound. In another embodiment, the second messenger responsecomprises a change in intracellular sodium ion levels and the change insecond messenger is a smaller increase in the measure of intracellularsodium ions. In another embodiment, the second messenger responsecomprises a change in intracellular potassium ion levels and the changein second messenger is a smaller increase in the measure ofintracellular potassium ions.

In one embodiment, the second messenger response comprises activation ofMAP kinase and the change in second messenger response is a smallerincrease in the level of MAP kinase activation in the presence of boththe chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another embodiment,the second messenger response comprises change in cAMP levels and thechange in second messenger response is a smaller change in the level ofcAMP in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound. In another embodiment, the second messenger response comprisesrelease of arachidonic acid and the change in second messenger responseis an smaller increase in the level of arachidonic acid levels in thepresence of both the chemical compound and the second chemical compoundthan in the presence of only the second chemical compound. In a furtherembodiment, the second messenger response comprises GTPγS ligand bindingand the change in second messenger is a smaller increase in GTPγS ligandbinding in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.

This invention provides a process for determining whether a chemicalcompound specifically binds to and enhances activation of a mammalianOSGPR114 or OSGPR78 receptor, which comprises separately contactingcells producing a second messenger response and expressing on their cellsurface the mammalian OSGPR114 or OSGPR78 receptor, wherein such cellsdo not normally express the mammalian OSGPR114 or OSGPR78 receptor, withboth the chemical compound and a second chemical compound known toactivate the mammalian OSGPR114 or OSGPR78 receptor, and with only thesecond chemical compound, under conditions suitable for activation ofthe mammalian OSGPR114 or OSGPR78 receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a larger change in the second messenger response inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compoundindicating that the chemical compound enhances activation of themammalian OSGPR114 or OSGPR78 receptor. In one embodiment the chemicalcompound that enhances activation of the mammalian OSGPR114 or OSGPR78receptor is an allosteric or allotopic agonist.

In one embodiment, the second messenger response comprises chloridechannel activation and the change in second messenger response is agreater increase in the level of chloride current in the presence ofboth the chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another embodiment,the second messenger response comprises change in intracellular calciumlevels and the change in second messenger response is a larger increasein the measure of intracellular calcium in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound. In another embodiment, the secondmessenger response comprises release of inositol phosphate and thechange in second messenger response is a larger increase in the level ofinositol phosphate in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound. In another embodiment, the second messenger responsecomprises a change in intracellular sodium ion levels and the change insecond messenger is a larger increase in the measure of intracellularsodium ions. In another embodiment, the second messenger responsecomprises a change in intracellular potassium ion levels and the changein second messenger is a larger increase in the measure of intracellularpotassium ions.

In one embodiment, the second messenger response comprises activation ofMAP kinase and the change in second messenger response is a largerincrease in the level of MAP kinase activation in the presence of boththe chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another embodiment,the second messenger response comprises change in cAMP levels and thechange in second messenger response is a larger change in the level ofcAMP in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound. In another embodiment, the second messenger response comprisesrelease of arachidonic acid and the change in second messenger responseis a larger increase in the level of arachidonic acid levels in thepresence of both the chemical compound and the second chemical compoundthan in the presence of only the second chemical compound. In a furtherembodiment, the second messenger response comprises GTPγS ligand bindingand the change in second messenger is a larger increase in GTPγS ligandbinding in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.

In one embodiment of the above processes the second messenger responseis measured by a change in reporter gene activity. Examples of reportergene include, but are not limited to, secreted alkaline phosphatase,luciferase, and β-galactosidase. Commonly used promoters In mammaliansystems examples of promoters that can be used with such reporter genesinclude, but are not limited to, CREB-responsive, NFAT-responsive,NFkB-responsive, SRE-responsive, and CyclinD1 promoters

In one embodiment, the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In a further embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a rat OSGPR114 or OSGPR78 receptor. Inanother embodiment, the mammalian OSGPR114 or OSGPR78 receptor is amouse OSGPR114 or OSGPR78 receptor. In another embodiment, the cell isan insect cell. In another embodiment, the cell is a yeast cell. Inanother embodiment, the cell is a mammalian cell. In another embodiment,the mammalian cell is a CHO, RH7777, Jurkat, HCT4, RBL243, or COS-7cell, a 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk−) cell.In another embodiment, the compound is not previously known to bind to amammalian OSGPR114 or OSGPR78 receptor.

In a preferred embodiment of the above processes of the invention, thesecond compound is an LPA compound. In an alternative embodiment thesecond compound is any of the compounds described in WO 02/29001; US2003/0027800 A1; U.S. Pat. No. 6,380,177; Hasegawa, Y., et al. (2003) J.Biol. Chem. 278(14):11962-9; Heise, C. E., et al. (2001) Mol. Pharmacol60(6):1173-80; Hooks, S. B., et al. (2001) J Biol. Chem. 276(7):4611-21;Hopper, D. W., et al. (1999) J. Med. Chem. 42(6):963-70; Tigyi, G.(2001) Mol Pharmacol. 60(6):1161-4; Yokoyama, K., et al. (2002).Biochim. Biophys. Acta 1582(1-3): 295-308; Gueguen, G., et al. (1999).Biochemistry 38(26): 8440-50; Lynch, K. R. and T. L. Macdonald (2002).Biochim. Biophys. Acta 1582(1-3): 289-94; Sardar, V. M., et al. (2002)Biochim. Biophys. Acta 1582: 309-307 or Virag, T., et al. (2003) MolPharmacol. 63(5):1032-42 as compounds that activate edg or LPAreceptors. In a further embodiment, LPA is selected from myristoyllysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid, or afunctional analog or homolog of one of these compounds. In analternative embodiment the lysophosphatidic acid is selected from1-myristoyl lysophosphatidic acid, 1-oleoyl lysophosphatidic acid,1-palmitoyl lysophosphatidic acid, and 1-stearoyl lysophosphatidic acid.

This invention provides a compound determined by a process or methodaccording to this invention and a composition, for example, apharmaceutical composition, which comprises an amount of a mammalianOSGPR114 or OSGPR78 receptor agonist determined to be such by a processaccording to this invention effective to increase activity of themammalian OSGPR114 or OSGPR78 receptor and a carrier, for example, apharmaceutically acceptable carrier. In one embodiment, the mammalianOSGPR114 or OSGPR78 receptor agonist is not previously known.

This invention provides a compound determined by a process or methodaccording to this invention and a composition, for example, apharmaceutical composition, which comprises an amount of a mammalianOSGPR114 or OSGPR78 receptor antagonist determined to be such by aprocess according to this invention, effective to reduce activity of themammalian OSGPR114 or OSGPR78 receptor and a carrier, for example, apharmaceutically acceptable carrier. In one embodiment, the mammalianOSGPR114 or OSGPR78 receptor antagonist is not previously known.

The compounds determined by a process or method according to thisinvention may be selective modulators of OSGPR114 or OSGPR78 activity,may be selective modulators of OSGPR114 and OSGPR78 activity but notactive on other LPA receptors, may be selective modulators of OSGPR114and/or OSGPR78 activity and active on a limited number of other LPAreceptors (e.g. one, two or three), or may be modulators of all LPAreceptors, in the latter case not necessarily with equivalent potency.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a mammalian OSGPR114 or OSGPR78 receptorto identify a compound which activates the mammalian OSGPR114 or OSGPR78receptor which comprises: (a) contacting cells transfected with andexpressing the mammalian OSGPR114 or OSGPR78 receptor with the pluralityof compounds not known to activate the mammalian OSGPR114 or OSGPR78receptor, under conditions permitting activation of the mammalianOSGPR114 or OSGPR78 receptor; (b) determining whether the activity ofthe mammalian OSGPR114 or OSGPR78 receptor is increased in the presenceof one or more of the compounds; and if so (c) separately determiningwhether the activation of the mammalian OSGPR114 or OSGPR78 receptor isincreased by any compound included in the plurality of compounds, so asto thereby identify each compound which activates the mammalian OSGPR114or OSGPR78 receptor. In one embodiment, the mammalian OSGPR114 orOSGPR78 receptor is a human OSGPR114 or OSGPR78 receptor. In a furtherembodiment, the mammalian OSGPR114 or OSGPR78 receptor is a rat OSGPR114or OSGPR78 receptor. In another embodiment, the mammalian OSGPR114 orOSGPR78 receptor is a mouse OSGPR114 or OSGPR78 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian OSGPR114 orOSGPR78 receptor to identify a compound which inhibits the activation ofthe mammalian OSGPR114 or OSGPR78 receptor, which comprises: (a)contacting cells transfected with and expressing the mammalian OSGPR114or OSGPR78 receptor with the plurality of compounds in the presence of aknown mammalian OSGPR114 or OSGPR78 receptor agonist, under conditionspermitting activation of the mammalian OSGPR114 or OSGPR78 receptor; (b)determining whether the extent or amount of activation of the mammalianOSGPR114 or OSGPR78 receptor is reduced in the presence of one or moreof the compounds, relative to the extent or amount of activation of themammalian OSGPR114 or OSGPR78 receptor in the absence of such one ormore compounds; and if so (c) separately determining whether each suchcompound inhibits activation of the mammalian OSGPR114 or OSGPR78receptor for each compound included in the plurality of compounds, so asto thereby identify any compound included in such plurality of compoundswhich inhibits the activation of the mammalian OSGPR114 or OSGPR78receptor.

In one embodiment, the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In a further embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a rat OSGPR114 or OSGPR78 receptor. Inanother embodiment, the mammalian OSGPR114 or OSGPR78 receptor is amouse OSGPR114 or OSGPR78 receptor. In another embodiment, the cell is ayeast cell. In another embodiment, the cell is a mammalian cell. Inanother embodiment, the mammalian cell is an adipose, breast, ovarian,colon, lung or liver cell. In another embodiment, the cell is a CHO,RH7777, Jurkat, HCT4, RBL243, or COS-7 cell, a 293 human embryonickidney cell, a LM(tk−) cell, a CHO cell, a mouse Y1 cell, or an NIH-3T3cell.

This invention provides a composition, for example a pharmaceuticalcomposition, comprising a compound identified by a method according tothis invention in an amount effective to increase mammalian OSGPR114 orOSGPR78 receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

This invention provides a composition, for example, a pharmaceuticalcomposition, comprising a compound identified by a method according tothis invention in an amount effective to decrease mammalian OSGPR114 orOSGPR78 receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by increasing the activity of amammalian OSGPR114 or OSGPR78 receptor which comprises administering tothe subject a compound which is a mammalian OSGPR114 or OSGPR78 receptoragonist in an amount effective to treat the abnormality. In oneembodiment, the abnormality is a regulation of a steroid hormonedisorder, an epinephrine release disorder, a gastrointestinal disorder,a cardiovascular disorder, an electrolyte balance disorder,hypertension, diabetes, a respiratory disorder, asthma, a reproductivefunction disorder, an immune disorder, an endocrine disorder, amusculoskeletal disorder, a neuroendocrine disorder, a cognitivedisorder, a memory disorder, somatosensory and neurotransmissiondisorders, a motor coordination disorder, a sensory integrationdisorder, a motor integration disorder, a dopaminergic functiondisorder, an appetite disorder, obesity, a sensory transmissiondisorder, an olfaction disorder, an autonomic nervous system disorder,pain, psychotic behavior, affective disorder, migraine, cancer,proliferative diseases, wound healing, tissue regeneration, bloodcoagulation-related disorders, developmental disorders, orischemia-reperfusion injury-related diseases.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of amammalian OSGPR114 or OSGPR78 receptor which comprises administering tothe subject a compound which is a mammalian OSGPR114 or OSGPR78 receptorantagonist in an amount effective to treat the abnormality. In oneembodiment, the abnormality is a regulation of a steroid hormonedisorder, an epinephrine release disorder, a gastrointestinal disorder,a cardiovascular disorder, an electrolyte balance disorder,hypertension, diabetes, a respiratory disorder, asthma, a reproductivefunction disorder, an immune disorder, an endocrine disorder, amusculoskeletal disorder, a neuroendocrine disorder, a cognitivedisorder, a memory disorder, somatosensory and neurotransmissiondisorders, a motor coordination disorder, a sensory integrationdisorder, a motor integration disorder, a dopaminergic functiondisorder, an appetite disorder, obesity, a somatosensoryneurotransmission disorder, an olfaction disorder, an autonomic nervoussystem disorder, pain, psychotic behavior, affective disorder, migraine,cancer, proliferative diseases, wound healing, tissue regeneration,blood coagulation-related disorders, developmental disorders, orischemia-reperfusion injury-related diseases.

This invention provides a process for making a composition of matterwhich specifically binds to a mammalian OSGPR114 or OSGPR78 receptorwhich comprises identifying a chemical compound using a process inaccordance with this invention and then synthesizing the chemicalcompound or a novel structural and functional analog or homolog thereof.In one embodiment, the mammalian OSGPR114 or OSGPR78 receptor is a humanOSGPR114 or OSGPR78 receptor. In another embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a rat OSGPR114 or OSGPR78 receptor. Inanother embodiment, the mammalian OSGPR114 or OSGPR78 receptor is amouse OSGPR114 or OSGPR78 receptor.

This invention provides a process for preparing a composition, forexample, a pharmaceutical composition which comprises admixing acarrier, for example, a pharmaceutically acceptable carrier, and apharmaceutically effective amount of a chemical compound identified by aprocess in accordance with this invention or a novel structural andfunctional analog or homolog thereof. In one embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a human OSGPR114 or OSGPR78 receptor. Inanother embodiment, the mammalian OSGPR114 or OSGPR78 receptor is a ratOSGPR114 or OSGPR78 receptor. In another embodiment, the mammalianOSGPR114 or OSGPR78 receptor is a mouse OSGPR114 or OSGPR78 receptor.

Thus, once the gene for a targeted receptor subtype such as OSGPR114 orOSGPR78 is cloned, it is placed into a recipient cell which thenexpresses the targeted receptor subtype on its surface. This cell, whichexpresses a single population of the targeted human receptor subtype, isthen propagated resulting in the establishment of a cell line. This cellline, which constitutes a drug discovery system, is used in twodifferent types of assays: binding assays and functional assays. Inbinding assays, the affinity of a compound for both the receptor subtypethat is the target of a particular drug discovery program and otherreceptor subtypes that could be associated with side effects aremeasured. These measurements enable one to predict the potency of acompound, as well as the degree of selectivity that the compound has forthe targeted receptor subtype over other receptor subtypes. The dataobtained from binding assays also enable chemists to design compoundstoward or away from one or more of the relevant subtypes, asappropriate, for optimal therapeutic efficacy. In functional assays, thenature of the response of the receptor subtype to the compound isdetermined. Data from the functional assays show whether the compound isacting to inhibit or enhance the activity of the receptor subtype, thusenabling pharmacologists to evaluate compounds rapidly at their ultimatehuman receptor subtypes targets permitting chemists to rationally designdrugs that will be more effective and have fewer or substantially lesssevere side effects than existing drugs.

Approaches to designing and synthesizing receptor subtype-selectivecompounds are well known and include traditional medicinal chemistry andthe newer technology of combinatorial chemistry, both of which aresupported by computer-assisted molecular modeling. With such approaches,chemists and pharmacologists use their knowledge of the structures ofthe targeted receptor subtype and compounds determined to bind and/oractivate or inhibit activation of the receptor subtype to design andsynthesize structures that will have activity at these receptorsubtypes.

Combinatorial chemistry involves automated synthesis of a variety ofnovel compounds by assembling them using different combinations ofchemical building blocks. The use of combinatorial chemistry greatlyaccelerates the process of generating compounds. The resulting arrays ofcompounds are called libraries and are used to screen for compounds(“lead compounds”) that demonstrate a sufficient level of activity atreceptors of interest. Using combinatorial chemistry it is possible tosynthesize “focused” libraries of compounds anticipated to be highlybiased toward the receptor target of interest.

Once lead compounds are identified, whether through the use ofcombinatorial chemistry or traditional medicinal chemistry or otherwise,a variety of homologs and analogs are prepared to facilitate anunderstanding of the relationship between chemical structure andbiological or functional activity. These studies define structureactivity relationships which are then used to design drugs with improvedpotency, selectivity and pharmacokinetic properties. Combinatorialchemistry is also used to rapidly generate a variety of structures forlead optimization. Traditional medicinal chemistry, which involves thesynthesis of compounds one at a time, is also used for furtherrefinement and to generate compounds not accessible by automatedtechniques. Once such drugs are defined the production is scaled upusing standard chemical manufacturing methodologies utilized throughoutthe pharmaceutical and chemistry industry.

In one aspect of this invention, OSGPR114 or OSGPR78 receptor activityis monitored by measuring a Gi/o-coupled readout. Gi/o-coupled readoutcan be monitored using an electrophysiological method to determine theactivity of G-protein regulated Ca²⁺ or K⁺ channels or by usingfluorescent dye to measure changes in intracellular Ca²⁺ levels. Othermethods that can typically be used to monitor receptor activity involvedmeasuring levels of or activity of GTPγS or cAMP.

Yeast assays may be used to screen for agents that modulate the activityof OSGPR114 or OSGPR78 or variant polypeptides. A typical yeast assayinvolves heterologously expressing OSGPR114 or OSGPR78 or a variantpolypeptide in a modified yeast strain containing multiple reportergenes, typically FUS1p-HIS-3 and FUS1p-lacZ, each linked to anendogenous MAPK cascade-based signal transduction pathway. This pathwayis normally linked to pheromone receptors, but can be coupled to foreignreceptors by replacement of the yeast G-protein with yeast/mammalianG-protein chimeras. Strains may also contain further gene deletions,such as deletions of SST2 and FAR1, to potentiate the assay. Ligandactivation of the heterologous receptor can be monitored for exampleeither as cell growth in the absence of histidine or with a suitablesubstrate for beta-galactosidase (lacZ). Such technology is well knownin the art. See for example WO 99/14344, WO 00/12704, or U.S. Pat. No.6,100,042.

Alternatively melanophore assays may be used to screen for modulators ofOSGPR114 or OSGPR78. OSGPR114 or OSGPR78 or a variant polypeptide can beheterologously expressed in Xenopus laevis melanophores and theiractivation or inhibition can be measured by either melanosome dispersionor aggregation. Basically, melanosome dispersion is promoted byactivation of adenylate cyclase or phospholipase C, i.e. Gs and Gqmediated signalling respectively, whereas aggregation results fromactivation of Gi-protein resulting in inhibition of adenylate cyclase.Hence, ligand activation of the heterologous receptor can be measuredsimply by measuring the change in light transmittance through the cellsor by imaging the cell response.

Preferably, control experiments are carried out on cells which do notexpress OSGPR114 or OSGPR78 or a variant polypeptide to establishwhether the observed responses are the result of activation of theOSGPR114 or OSGPR78 or the variant polypeptide.

Suitable test substances which can be tested in the above assays includecombinatorial libraries, defined chemical entities, peptide and peptidemimetics, oligonucleotides and natural product libraries, such asdisplay (e.g. phage display libraries) and antibody products. In oneembodiment, the test substance is an LPA compound or a closely relatedcompound. Assays may also be carried out using known ligands of otherG-protein coupled receptors to identify additional ligands which act asagonists of OSGPR114 or OSGPR78.

Test substances may be used in an initial screen of, for example, 10substances per reaction, and the substances of these batches which showinhibition or activation tested individually. Test substances may beused at a concentration of from 1 nM to 1000 μM, preferably from 1 μM to100 μM, more preferably from 1 μM to 10 μM.

Agents which modulate OSGPR114 or OSGPR78 activity and which have beenidentified by assays in accordance with the invention can be used in thetreatment or prophylaxis of diabetes, obesity or feeding disorders whichare responsive to regulation of OSGPR114 or OSGPR78 receptor activity,and are one embodiment of this invention

Agents which inhibit OSGPR114 or OSGPR78 receptor activity and/or whichhave been identified as inhibitors of cell proliferation are a preferredembodiment. In particular, such agents may be used in the treatment ofcertain cancers, including, but not limited to, ovarian cancer and othergynecological cancers, cancers of the lung, prostate, pancreas, colon,breast, esophagus, kidney and stomach, and glioma, lymphoma, leukemiaand melanoma, and also obesity, atherosclerosis, restenosis, renalischemia, and certain reproductive disorders.

In an alternative embodiment, agents which activate OSGPR114 or OSGPR78receptor activity and/or which have been identified as stimulators ofcell proliferation are also useful. In particular, such agents may beused in the treatment of wound healing and immune system disordersrequiring activation of immune system cells (e.g. T-cells).

The amount of a OSGPR114 or OSGPR78 modulator which is required toachieve the desired biological effect will, of course, depend on anumber of factors, for example, the mode of administration and theprecise clinical condition of the recipient. In general, the daily dosewill be in the range of 0.1-1000 mg/kg, typically 0.1-100 mg/kg. Anintravenous dose may, for example, be in the range of 0.01 mg to 100mg/kg, typically 0.01 to 10 mg/kg, which may conveniently beadministered as an infusion of from 0.1 μg to 1 mg, per minute. Infusionfluids suitable for this purpose may contain, for example, from 0.01 μgto 0.1 mg, per milliliter. Unit doses may contain, for example, from0.01 μg to 1 g of a OSGPR114 or OSGPR78 modulator. Thus ampoules forinjection may contain, for example, from 0.01 μg to 0.1 g and orallyadministrable unit dose formulations, such as tablets or capsules, maycontain, for example, from 0.1 mg to 1 g.

An OSGPR114 or OSGPR78 modulator may be employed in the treatment of aOSGPR114 or OSGPR78 mediated disease as the compound per se, but ispreferably presented with an acceptable carrier in the form of apharmaceutical formulation. The carrier must, of course, be acceptablein the sense of being compatible with the other ingredients of theformulation and must not be deleterious to the recipient. The carriermay be a solid or a liquid, or both, and is preferably formulated withthe OSGPR114 or OSGPR78 modulator as a unit-dose formulation, forexample, a tablet, which may contain from 0.05% to 95% by weight of theOSGPR114 or OSGPR78 modulator.

The formulations include those suitable for oral, rectal, topical,buccal (e.g. sub-lingual) and parenteral (e.g. subcutaneous,intramuscular, intradermal or intravenous) administration. Formulationssuitable for oral administration may be presented in discrete units,such as capsules, cachets, lozenges or tablets, each containing apredetermined amount of a OSGPR114 or OSGPR78 modulator; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. In general, theformulations are prepared by uniformly and intimately admixing theactive OSGPR114 or OSGPR78 modulator with a liquid or finely dividedsolid carrier, or both, and then, if necessary, shaping the product. Forexample, a tablet may be prepared by compressing or moulding a powder orgranules of the OSGPR114 or OSGPR78 modulator optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the compound in a free-flowing form,such as a powder or granules optionally mixed with a binder, lubricant,inert diluent and/or surface active/dispersing agent(s). Moulded tabletsmay be made by moulding, in a suitable machine, the powdered compoundmoistened with an inert liquid diluent.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising a OSGPR114 or OSGPR78 modulator in a flavoured base,usually sucrose and acacia or tragacanth, and pastilles comprising theOSGPR114 or OSGPR78 modulator in an inert base such as gelatin andglycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of anOSGPR114 or OSGPR78 modulator, preferably isotonic with the blood of theintended recipient. These preparations are preferably administeredintravenously, although administration may also be effected by means ofsubcutaneous, intramuscular, or intradermal injection. Such preparationsmay conveniently be prepared by admixing the OSGPR114 or OSGPR78modulator with water and rendering the resulting solution sterile andisotonic with the blood. Injectable compositions according to theinvention will generally contain from 0.1 to 5% w/w of the OSGPR114 orOSGPR78 modulator.

Formulations suitable for rectal administration are preferably presentedas unit-dose suppositories. These may be prepared by admixing a OSGPR114or OSGPR78 modulator with one or more conventional solid carriers, forexample, cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanolin,polyethylene glycols, alcohols, and combinations of two or more thereof.The OSGPR114 or OSGPR78 modulator is generally present at aconcentration of from 0.1 to 15% w/w of the composition, for example,from 0.5 to 2%.

Alternatively, agents which up-regulate OSGPR114 or OSGPR78 expressionor nucleic acids encoding OSGPR114 or OSGPR78 or a variant polypeptidemay be administered to the mammal. Nucleic acid, such as RNA or DNA,preferably DNA, is provided in the form of a vector, which may beexpressed in the cells of a human or other mammal under treatment.Preferably such up-regulation or expression following nucleic acidadministration will enhance OSGPR114 or OSGPR78 activity.

OSGPR114 or OSGPR78 antisense nucleic acid or RNAi may also be used todecrease OSGPR114 or OSGPR78 protein levels and thus OSGPR114 or OSGPR78activity, for use in cell proliferation disorders requiring inhibitionof cell proliferation (e.g. cancer), or other OSGPR114 or OSGPR78mediated diseases that would benefit from a decrease in OSGPR114 orOSGPR78 activity.

Nucleic acid encoding OSGPR114 or OSGPR78 or variant polypeptide may beadministered to a human or other mammal by any available technique. Forexample, the nucleic acid may be introduced by injection, preferablyintradermally, subcutaneously or intramuscularly. Alternatively, thenucleic acid may be delivered directly across the skin using a nucleicacid delivery device such as particle-mediated gene delivery. Thenucleic acid may be administered topically to the skin, or to themucosal surfaces for example by intranasal, oral, intravaginal,intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several knowntransfection techniques, for example those including the use oftransfection agents. Examples of these agents include cationic agents,for example, calcium phosphate and DEAE-dextran and lipofectants, forexample, lipofectam and transfectam. The dosage of the nucleic acid tobe administered can be altered. Typically the nucleic acid isadministered in the range of 1 pg to 1 mg, preferably to 1 pg to 10 μgnucleic acid for particle mediated gene delivery and 10 μg to 1 mg forother routes.

Polynucleotides encoding OSGPR114 or OSGPR78 or a variant polypeptidecan also be used to identify mutation(s) in OSGPR114 or OSGPR78 geneswhich may be implicated in human disorders. Identification of suchmutation(s) may be used to assist in diagnosis of feeding disorders andconditions associated with feeding disorders such as obesity, type IIdiabetes, insulin resistance and metabolic syndrome (syndrome X), orsusceptibility to such disorders and in assessing the physiology of suchdisorders.

Antibodies (either polyclonal or preferably monoclonal antibodies,chimeric, single chain, Fab fragments) which are specific for theOSGPR114 or OSGPR78 polypeptides or a variant thereof can be generated.Such antibodies may for example be useful in purification, isolation orscreening methods involving immunoprecipitation techniques and may beused as tools to elucidate further the function of OSGPR114 or OSGPR78or a variant thereof, or indeed as therapeutic agents in their ownright. Such antibodies may be used to block ligand binding to thereceptors. A variety of protocols for competitive binding orimmunoradiometric assays to determine the specific binding capability ofan antibody are well known in the art (see for example Maddox et. al, J.Exp. Med. 158: 1211, 1993).

The activators, inhibitors, polynucleotides and antibodies for use inthe instant invention may be used in combination with one or more othertherapeutic agents. In one embodiment, in the treatment of cancer, otheranticancer agents may be used in combination with an OSGPR114 or OSGPR78receptor inhibitor, including, but not limited to alkylating agents,such as cyclophosphamide (CTX; cytoxan), chlorambucil (CHL; leukeran),cisplatin (CisP; platinol) busulfan (myleran), melphalan, carmustine(BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and thelike alkylating agents; anti-metabolites, such as methotrexate (MTX),etoposide (VP16; vepesid) 6-mercaptopurine (6MP), 6-thiocguanine (6TG),cytarabine (Ara-C), 5-fluorouracil (5FU), dacarbazine (DTIC), and thelike anti-metabolites; antibiotics, such as actinomycin D, doxorubicin(DXR; adriamycin), daunorubicin (daunomycin), bleomycin, mithramycin andthe like antibiotics; alkaloids, such as vinca alkaloids such asvincristine (VCR), vinblastine, and the like; and other antitumoragents, such as taxol and taxol derivatives, the cytostatic agents,glucocorticoids such as dexamethasone (DEX; decadron) andcorticosteroids such as prednisone, nucleoside enzyme inhibitors such ashydroxyurea, amino acid depleting enzymes such as asparaginase, andsimilar, diverse antitumor agents.

The use of the cytotoxic agents described above in chemotherapeuticregimens is generally well characterized in the cancer therapy arts, andtheir use herein falls under the same considerations for monitoringtolerance and effectiveness and for controlling administration routesand dosages, with some adjustments. For example, the actual dosages ofthe cytotoxic agents may vary depending upon the patient's cultured cellresponse determined by using the present histoculture methods.Generally, the dosage will be reduced compared to the amount used in theabsence of agents that affect OSGPR114 or OSGPR78.

Typical dosages of an effective cytotoxic agent can be in the rangesrecommended by the manufacturer, and where indicated by in vitroresponses or responses in animal models, can be reduced by up to aboutone order of magnitude concentration or amount. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based on the invitro responsiveness of the primary cultured malignant cells orhistocultured tissue sample, or the responses observed in theappropriate animal models.

In one embodiment of this invention a protein-tyrosine kinase inhibitormay be used as a therapeutic agent in combination with an OSGPR114 orOSGPR78 receptor inhibitor of this invention. The protein-tyrosinekinase inhibitor may be, for example, an EGFR inhibitor, an IGFRinhibitor, a src kinase inhibitor, a PDGFR inhibitor, a HER2 kinaseinhibitor, or an inhibitor of other protein-tyrosine kinases regulatedby LPA, or an inhibitor of other protein-tyrosine kinases that areinvolved in causing abnormal cell growth or cancer. In one aspect ofthis invention these combinations are used for the treatment of thefollowing disorders—cancers of the colon, lung, ovarian, breast,prostate, pancreas, esophagus, kidney, and stomach, and glioma,leukemia, lymphoma, and melanoma. In one embodiment the EGFR inhibitoris Tarceva (OSI-774), Iressa (gefinitib, ZD-1839) or an EGFR antibody(e.g. Erbitux (C225)).

In an alternative embodiment, in the treatment of obesity, Orlistat,Sibutramine, or a cannabinoid CB1 receptor antagonist may be used incombination with an OSGPR114 or OSGPR78 receptor modulator. Theinvention thus provides in a further aspect the use of a combination ofan OSGPR114 or OSGPR78 modulator and at least one other therapeuticagent in the treatment of OSGPR114 or OSGPR78 mediated disorders.

When the activators, inhibitors and polynucleotides and antibodies areused in combination with other therapeutic agents, the agents may beadministered either sequentially or simultaneously by any convenientroute.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above optimallytogether with a pharmaceutically acceptable carrier or excipientcomprise a further aspect of the invention. The individual components ofsuch combinations may be administered either sequentially orsimultaneously in separate or combined pharmaceutical formulations.

When combined in the same formulation it will be appreciated that thetwo components must be stable and compatible with each other and theother components of the formulation and may be formulated foradministration. When formulated separately they may be provided in anyconvenient formulation, conveniently in such a manner as are known forsuch compounds in the art.

When in combination with a second therapeutic agent active against thesame disease, the dose of each component may differ from that when thecompound is used alone. Appropriate doses will be readily appreciated bythose skilled in the art.

Compounds of the invention that are identified as modulators of OSGPR114or OSGPR78 activity can be screened by a variety of means known in theart to demonstrate their pharmacological activity. Compounds that haveanti-cancer or anti-tumor activity for instance, can be identified invivo using animal bioassay techniques known to those of ordinary skillin the art. Test compounds and appropriate vehicle controls can beadministered by any of a number of routes (e.g., the oral route, aparenteral route) to experimental subjects and the size and growth oftumors can be monitored over the course of therapy. The experimentalsubjects are humans or test animals (e.g., rats, mice).

The inhibitory effective amount of a compound can be determined usingart-recognized methods, such as by establishing dose response curves insuitable animal models and extrapolating to human; extrapolating fromsuitable in vitro data; or by determining effectiveness in clinicaltrials. Suitable doses of compounds of the invention depend upon theparticular medical application, such as the severity of the disease, theweight of the individual, age of the individual, half-life incirculation, etc., and can be determined readily by the skilled artisan.The number of doses, daily dosage and course of treatment may vary fromindividual to individual.

The effects of compounds that modulate OSGPR114 or OSGPR78 activity oncell proliferation can be monitored by any of the many methods known tothose of ordinary skill in the art, or modifications thereof (e.g. seeMethods section herein).

The effects of compounds that modulate OSGPR114 or OSGPR78 activity onapoptosis can be monitored by any of the many methods known to those ofordinary skill in the art, or modifications thereof. Specific examplesof apoptosis assays are exemplified in the following references. Assaysfor apoptosis in lymphocytes are disclosed by: Li et. al., (1995)Science 268:429-431; Gibellini et. al. (1995) Br. J. Haematol. 89:24-33;Martin et al. (1994) J. Immunol. 152:330-42; Terai et al., (1991) J.Clin Invest. 87:1710-5; Dhein et al. (1995) Nature 373:438-441; Katsikiset al. (1995) J. Exp. Med. 1815:2029-2036; Westendorp et al. (1995)Nature 375:497; and DeRossi et al. (1994) Virology 198:234-44. Assaysfor apoptosis in fibroblasts are disclosed by: Vossbeck et al. (1995)Int. J. Cancer 61:92-97; Goruppi et al. (1994) Oncogene 9:1537-44;Fernandez et. al. (1994) Oncogene 9:2009-17; Harrington et al. (1994)EMBO J., 13:3286-3295; and Itoh et al., (1993) J. Biol. Chem.268:10932-7. Assays for apoptosis in neuronal cells are disclosed by:Melino et al. (1994) Mol. Cell Biol. 14:6584-6596; Rosenblaum et al.(1994) Ann. Neurol. 36:864-870; Sato et. al. (1994) J. Neurobiol.25:1227-1234; Ferrari et al. (1995) J. Neurosci. 1516:2857-2866; Talleyet. al. (1995) Mol. Cell Biol. 1585:2359-2366; Talley et al. (1995) Mol.Cell. Biol. 15:2359-2366; and Waikinshaw et al. (1995) J. Clin. Invest.95:2458-2464. Assays for apoptosis in insect cells are disclosed by:Clem et al. (1991) Science 254:1388-90; Crook et al. (1993) J. Virol:67:2168-74; Rabizadeh et al. (1993) J. Neurochem. 61:2318-21; Birnbaumet al. (1994) J. Virol. 68:2521-8, 1994; and Clem et al. (1994) Mol.Cell. Biol. 14:5212-5222.

The effects of compounds that modulate OSGPR114 or OSGPR78 activity oncell motility, invasion and metastasis can be monitored by any of themany methods known to those of ordinary skill in the art, ormodifications thereof (e.g. see Methods section herein). A typical assayfor measuring cellular chemotaxis utilizes a so-called “Boyden chamber”(Boyden S. (1962) J. Exp. Med. 115:453-66), or similar apparatus, inwhich the cells migrate through a filter that has pore openings that aresmaller than the cell diameter. Typically, cells of a particular typeare placed in a chamber on one side of the filter and a chemotacticagent is placed in a chamber on the other side. Results are usuallyquantified by counting the number of cells that have migrated throughthe filter.

The effects of compounds that modulate OSGPR114 or OSGPR78 activity onangiogenesis or proangiogenic factor secretion by cells can be monitoredby any of the many methods known to those of ordinary skill in the art,or modifications thereof. Angiogenesis can be considered to be theresult of three distinct cellular activities: proliferation, migrationand differentiation. Assays exist which model each of these phasesseparately. Simple in vitro tests measure changes in proliferation of arange of cell types and assess migration over basement membraneproteins. Current in vitro assay systems, which depend on provision of aprotein matrix, effectively measure the ability of endothelial cells todifferentiate. Assay systems measuring differentiation involve theformation of cord-like structures by endothelial cells. All such systemsdepend on supplying the cells with exogenous basement membrane proteinson which the cells migrate to form tubules. Cell migration occurs overrelatively short time periods of 2-16 hours to give a three dimensionalstructure. In addition to the basement membrane proteins, many of thesystems require the provision of growth factors to produce acceptabletubule formation. The time scale over which tubules are formed providesan excellent test for inhibition of differentiation.

Other angiogenesis assays that measure the complete process are based onin vivo systems. Three frequently used systems are the rabbit cornealpocket, the hamster cheek pouch and the chicken chorioallantoic membrane(CAM) assays (Folkman, J. and Brem, H. (1992) Angiogenesis andinflammation. In: “Inflammation. Basic Principles and ClinicalCorrelates”. Eds Gallin, J. I., et. al., Raven Press, New York; Folkman,J. (1985) Adv. Cancer Res. 43:175; Folkman, J. and Klagsbrun, M. (1987)Science 235:442; Folkman, J. (1985) Perspect. Biol. Med. 29:10). In eachsystem an angiogenic substances is implanted in the cornea, cheek pouchor the CAM in order to induce angiogenesis. In all three assays asustained-release polymeric vehicle is used for delivery of theangiogenic substance and inhibitor compounds ((Langer, R and Folkman, J.(1976) Nature 263:797).

An alternative assay system (Yan, et al., (1993) J. Clin. Invest.,91:986-996) measures human angiogenesis, invasion and metastasis in achimeric mouse:human model, and is referred to as the experimental humanangiogenesis assay. It is a useful assay model for in vivo angiogenesisbecause the transplanted skin grafts closely resemble normal human skinhistologically. In this model, human cancer cell invasion andneovascularization are occurring wherein actual human blood vessels andtissue are growing from the grafted human skin into the human tumortissue on the surface of the grafted human skin. The origin of theneovascularization into the human graft can be demonstrated byimmunohistochemical staining of the neovasculature with human-specificendothelial cell markers. The invasion and metastasis of human cancercells may be monitored also. The assay can be used to demonstrateregression of neovascularization based on both the amount and extent ofregression of new vessel growth. Effects on the invasion and metastasisof any cancer tissue transplanted upon the grafted skin are easilymonitored. The assay is particularly useful because there is an internalcontrol for toxicity in the assay system. The SCID mouse is exposed toany test reagent, and therefore the health of the mouse is an indicationof toxicity.

The effects of compounds that modulate OSGPR114 or OSGPR78 activity ontumor growth can be monitored by any of the many methods known to thoseof ordinary skill in the art, or modifications thereof. For example, theefficacy of the compounds can be tested in animal models. For example,the efficacy of the compound alone or in combination with conventionalanti-tumor agents such as cytotoxic/anti-neoplastic agents andanti-angiogenic agents can be compared to the conventional agents alone.Typically, a tumor of a given size is present in a rat or mouse. Themouse is treated with the agent and the size of the tumor is measuredover time. The mean survival time of the animals can also be measured.The compounds modulating OSGPR114 or OSGPR78 receptor activity that areused must actively bind the receptor in the animal which is to betested. Xenografts can be implanted into the animal to test the abilityof species specific compounds to inhibit. Suitable test animals include,but are not limited to, inbred rats such as Fischer 344 and Lewis rats,and athymic NCR-NU mice.

Compounds of the invention that are identified as modulators of OSGPR114or OSGPR78 activity can be screened by a variety of means known in theart to demonstrate pharmacological activity in obesity. Body fatreducing compounds, for instance, can be identified in vivo using animalbioassay techniques known to those of ordinary skill in the art. Testcompounds and appropriate vehicle or caloric controls can beadministered by any of a number of routes (e.g., the oral route, aparenteral route) to experimental subjects and the weight of thesubjects can be monitored over the course of therapy. The experimentalsubjects are humans or test animals (e.g., rats, mice).

The effect of the compound on appetite or in inducing hypophagia orreduced food intake can be assessed, for instance, by monitoring thefood consumption of the test subjects (e.g., measuring the amount eatenor not eaten by a subject in terms of food weight or caloric content).The effect of the compounds on appetite can also be assessed bysubjective means including questionnaires as to appetite or foodcravings levels by human subjects. The effect of the test compounds onlipid metabolism can be assessed by monitoring blood lipids and fattyacid oxidation. The techniques for these assessments are well known tothose of ordinary skill in the art. The studies may be acute, subacute,chronic, or subchronic with respect to the duration of administrationand or follow-up of the effects of the administration.

Body fat reduction can be determined, for instance, by directlymeasuring changes in body fat of the animal or by measuring changes inthe body weight of the animal. The animal may be selected from the groupconsisting of a mouse, a rat, a guinea pig, or a rabbit. The animal mayalso be an ob/ob mouse, a db/db mouse, or a Zucker rat or other animalmodel for a weight-associated disease. Clinical studies in humans mayalso be conducted.

Compounds of the invention can be administered to an animal to determinewhether they affect food intake and body weight, body fat, appetite,food seeking behavior, or modulate fatty acid oxidation. Animals can be,for example, obese or normal guinea pigs, rats, mice, or rabbits.Suitable rats include, for example, Zucker rats. Suitable mice include,for example, normal mice, ALS/LtJ, C3.SW-H-^(2b)/Snj,(NON/LtJ×NZO/H1J)F1, NZO/H1J, ALR/LtJ, NON/LtJ, KK.Cg-AALR/LtJ, NON/LtJ,KK.Cg-A^(Y)/J, B6.HRS(BKS)-Cpe^(fat)/+, B6.129P2-Gck^(tm/Efr),B6.V-Lep^(ob), BKS.Cg-m +/+ Lep^(rd)b, and C57BL/6J with diet inducedobesity.

Administration of an appropriate amount the candidate compound may be byany means known in the art such as, for example, oral or rectal,parenteral such as, for example, intraperitoneal, intravenous,subcutaneous, subdermal, intranasal, or intramuscular. Preferablyadministration may be intraperitoneal or oral. An appropriate effectiveamount of the candidate compound may be determined empirically as isknown in the art. An appropriate effective amount may be an amountsufficient to effect a loss of body fat or a loss in body weight orreduction in food consumption in the animal over time. The candidatecompound can be administered as often as required to effect a loss ofbody fat or loss in body weight, for example, hourly, every six, eight,twelve, or eighteen hours, daily, or weekly. Formulations suitable fororal administration include (a) liquid solutions, such as an effectiveamount of the candidate compound suspended in diluents, such as water,saline or PEG 400; (b) capsules, sachets or tablets, each containing apredetermined amount of the active ingredient, as liquids, solids,granules or gelatin; (c) suspensions in an appropriate liquid; and (d)suitable emulsions. Tablet forms include one or more of lactose,sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potatostarch, microcrystalline cellulose, gelatin, colloidal silicon dioxide,talc, magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, e.g., sucrose, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, carriers known in the art.

Injection, solutions, and suspensions, can be prepared from sterilepowders, granules, and tablets of the kind previously described.Formulations suitable for parenteral administration, include, forexample, aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives.

The dose administered to the animal is sufficient to effect a change inbody weight, body fat, and/or fatty acid oxidation over time. Such adose can be determined according to the efficacy of the particularcandidate compound employed and the condition of the animal, as well asthe body weight or surface area of the animal. The size of the dose alsowill be determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a candidate compound;the LD₅₀ of the candidate compound; and the side-effects of thecandidate compound at various concentrations. In general, the dose willrange from 0.1-50 mg per kg, preferably 1-25 mg per kg, most preferably1-20 mg per kg body weight. The determination of dose responserelationships is well known to one of ordinary skill in the art.

Body weight reduction is typically determined by direct measurements ofthe change in body fat or by loss of body weight. Body fat and bodyweight of the animals is determined before, during, and after theadministration of the candidate compound. Changes in body fat aremeasured by any means known in the art such as, for example, fat foldmeasurements with calipers, bioelectrical impedance, hydrostaticweighing, or dual x-ray absorbiometry (e.g. DEXA). Preferably animalsdemonstrate at least 2%, 5%, 8%, or 10% loss of body fat. Changes inbody weight can be measured by any means known in the art such as, forexample, on a portable scale, on a digital scale, on a balance scale, ona floor scale, or a table scale. Preferably animals demonstrate at least2%, 5%, 10%, or 15% loss of body weight. Body weight reduction ismeasured before administration of the candidate compound and at regularintervals during and after treatment. Preferably, body weight ismeasured every 5 days, more preferably every 4 days, even morepreferably every 3 days, yet more preferably every 2 days, mostpreferably every day.

Changes in fatty acid metabolism can be measured by looking at fattyacid oxidation in cells from major fat burning tissues such as, forexample, liver (Beynen, et al. Diabetes 28:828 (1979)), muscle (ChiassonLab. Anat. of Rat, (1980)), heart (Flink, et al. J Biol. Chem. 267: 9917(1992)), and adipocytes (Rodbell, J. Biol. Chem. 239: 375 (1964)), Cellsmay be from primary cultures or from cell lines. Cells may be preparedfor primary cultures by any means known in the art including, forexample, enzymatic digestion and dissection. Suitable cell lines areknown to those in the art. Suitable hepatocyte lines are, for example,Fao, MH1C1, H-4-II-E, H4TG, H4-II-E-C3, McA-RH7777, McA-RH8994, N1-S1Fudr, N1-S1, ARL-6, Hepa 1-6, Hepa-1c1c7, BpRc1, tao BpRc1, NCTC clone1469, PLC/PRF/5, Hep 3B2.1-7 [Hep 3B], Hep G2 [HepG2], SK-HEP-1, andWCH-17. Suitable skeletal muscle cell lines are, for example, L6, L8,C8, NOR-10, BLO-11, BC3H1, G-7, G-8, C2C12, P19, SoI8, SJRH30 [RMS 13],and QM7. Suitable cardiac cell lines are, for example, H9c2(2-1), P19,CCD-32Lu, CCD-32Sk, Girardi, and FBHE. Suitable adipocyte lines are, forexample, NCTC clone 929 [derivative of Strain L; L-929; L cell], NCTC2071, L-M, L-M(TK−) [LMTK−; LM(tk−)], A9 (APRT and HPRT negativederivative of Strain L), NCTC clone 2472, NCTC clone 2555, 3T3-L1, J26,J27-neo, J27-B7, MTKP 97-12 pMp97b [TKMp97-12], L-NGC-5HT2, Ltk-11,L-alpha-1b, L-alpha-2A, L-alpha-K, and B82.

The rate of fatty acid oxidation may be measured by ¹⁴C-oleate oxidationto ketone bodies (Guzman and Geelen, Biochem. J. 287:487 (1982)) and/or¹⁴C-oleate oxidation to CO₂ (Fruebis, PNAS 98:2005 (2001); Blazquez et.al., J Neurochem 71:1597 (1998)). Lipolysis may be measured by fattyacid or glycerol release by using appropriate labeled precursors orspectrophotometric assays (Serradeil-Le Gal, FEBS Lett., 475:150(2000)). For analysis of ¹⁴C-oleate oxidation to ketone bodies, freshlyisolated cells or cultured cell lines can be incubated with ¹⁴C-oleicacid for an appropriate time, such as, for example, 30, 60, 90, 120, or180 minutes. The amount of ¹⁴C radioactivity in the incubation mediumcan be measured to determine their rate of oleate oxidation. Oleateoxidation can be expressed as nmol oleate produced in x minutes per gcells. For analysis of lypolysis/glycerol release, freshly isolatedcells or cultured cells lines can be washed then incubated for anappropriate time. The amount of glycerol released into the incubationmedia can provide an index for lipolysis.

Many alternative experimental methods known in the art may besuccessfully substituted for those specifically described herein in thepractice of this invention, as for example described in many of theexcellent manuals and textbooks available in the areas of technologyrelevant to this invention (e.g. Using Antibodies, A Laboratory Manual,edited by Harlow, E. and Lane, D., 1999, Cold Spring Harbor LaboratoryPress, (e.g. ISBN 0-87969-544-7); Roe B. A. et. al. 1996, DNA Isolationand Sequencing (Essential Techniques Series), John Wiley & Sons. (e.g.ISBN 0-471-97324-0); Methods in Enzymology: Chimeric Genes andProteins”, 2000, ed. J. Abelson, M. Simon, S. Emr, J. Thorner. AcademicPress; Molecular Cloning: a Laboratory Manual, 2001, 3^(rd) Edition, byJoseph Sambrook and Peter MacCallum, (the former Maniatis Cloningmanual) (e.g. ISBN 0-87969-577-3); Current Protocols in MolecularBiology, Ed. Fred M. Ausubel, et. al. John Wiley & Sons (e.g. ISBN0-471-50338-X); Current Protocols in Protein Science, Ed. John E.Coligan, John Wiley & Sons (e.g. ISBN 0-471-11184-8); and Methods inEnzymology: Guide to protein Purification, 1990, Vol. 182, Ed.Deutscher, M. P., Academic Press, Inc. (e.g. ISBN 0-12-213585-7)), or asdescribed in the many university and commercial websites devoted todescribing experimental methods in molecular biology.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter, and are not to be considered in any way limited thereto.

EXPERIMENTAL DETAILS Materials and Methods

Lipid Compounds

All LPA compounds used in the experiments described herein werepurchased from Sigma-Aldrich (USA) or Avanti Polar Lipids (Alabaster,Ala., USA), and were all of sn1 configuration. LPA compounds can also besynthesized by methods known in the art (e.g. see Bandoh, K. et. al.(2000) FEBS Letters 478:158-165, and references cited therein).

Cell Lines and Reagents

Cell lines were purchased from the American Type Culture Collection andmaintained in a suitable growth medium (e.g. DMEM) supplemented withFetal Bovine Serum (e.g. 10% FBS). pcDNA 3.1 mammalian expressionvector, PCR Blunt cloning vector, and DH5α competent E. coli cells werepurchased from Invitrogen Life Corporation. PCR reagents were from RocheMolecular Systems, Inc. (N808-0228). Restriction endonucleases were fromNew England Biolabs, Inc. The primers used for PCR were synthesized byACGT, Inc. Transfection reagents were purchased from Roche.

Cloning of Human and Mouse OSGPR114 or OSGPR78 Receptors

The OSGPR114 and OSGPR78 human and mouse genes are single exon genes ofapproximately 1.1 and 1.0 kilobase pairs in length with a 67.91% and35.94% GC content respectively. The receptor genes were PCR amplifiedfrom 0.1 g human genomic DNA samples in a 25 μl reaction volume usingthe appropriate oligonucleotides 1-4 (FIG. 11) at 0.5 μM each, 0.5 mMdNTP mix, 1.5×Pfu buffer, and 2.5 units Pfu-turbo enzyme (Stratagene).PCR cycling conditions were set as follows: 2 minute hold at 95° C.; 35cycles of template denaturing 15 seconds at 95° C., primer annealing 30seconds at 55° C., primer extension 1 minute 30 seconds at 72° C.; 7minute hold at 72° C.; final hold at 4° C. PCR DNA was purified from PCRreaction mix according to QIAGEN's QIAquick PCR purification spin columnprotocol.

Resulting DNA fragments were consecutively digested in a 1001 finalreaction volume with appropriate restriction enzymes (1 unit/μg DNA)using manufacturers defined conditions for optimal enzyme use. Theoligonucleotides were designed to create an NcoI restriction site at thereceptor gene's ATG initiation codon. Following fragment digestion, DNAwas gel purified from a 1.5% agarose gel using a QIAquick gel extractionprotocol (QIAGEN).

The OSGPR114 and OSGPR78 genes were cloned into a yeast expressionvector at the NcoI/XbaI restriction sites in a (cohesive) sticky-endligation reaction. Ligation reactions were set up at a 3:1 insert tovector ratio using 25 ng digested parent yeast expression vector. The 20μl reaction mix containing the DNA, 1× ligase buffer, and 20 units T4DNA ligase, was incubated at room temperature for 1 hour. In theresulting construct, GPCR expression is under the control of the PGKpromoter while the N-terminus of the receptor gene is fused to an 89amino acid Mfα1-Leader sequence. Mfα1 is responsible for transportingthe receptor to the cell membrane and is crucial for the performance ofthe FUS1p-LacZ yeast assay.

The receptor-vector was then transformed into TOPTEN chemicallycompetent E. coli cells (Invitrogen). Between 1 μl and 5 μl ligationreaction product was introduced to 50 μl cells and incubated on ice for30 minutes. Cells were then heat shocked for 30 seconds at 42° C.,followed by 1 hour incubation in 250 μl S.O.C. media at 37° C. withagitation (225 rpm). After the 1 hour growth period, cells were spreadout on LB agar plates with ampicillin and incubated overnight at 37° C.E. coli transformants containing ampicillin resistance were picked thefollowing day and set up for plasmid isolation according to QIAGEN'sminiprep protocol. DNA was analyzed by restriction digest using enzymecombination BglII/BamHI. Miniprep DNA digests resulting in a 7.6 kb+1.2kb banding pattern on an agarose gel confirmed which plasmids containedthe OSGPR114/OSGPR78 gene. Sequencing analysis of several clones wasconducted. In a BLAST pairwise sequence alignment, the resultingsequence data from two individual DNA preparations were found to be 100%identical with the OSGPR114 sequence found in the literature. Twodistinct sequences were identified from the samples of human genomic DNAfor the OSGPR78 gene, one of which was consistent with the publishedsequence of OSGPR78 and one that contained a conservative Valine toIsoleucine amino acid substitution at position 33. It is feasible thatthis may reflect a polymorphism of the receptor, as the same change wasobserved in different PCR reactions from the same sample.

OSGPR114 and OSGPR78 were subcloned into pcDNA3.1, zeo mammalianexpression vector from the yeast expression vectors by EcoRI/BamHI (forOSGPR114) or HindIII/Xho (for OSGPR78) digestion of the parent plasmidand pcDNA plasmid and the fragment from the yeast expression vectordigestion ligated to the pcDNA3.1 digestion.

The rat, mouse, dog, or other mammalian homologs of OSGPR114 or OSGPR78receptor sequences can readily be cloned by comparable methods.

Host Cells for Expression of Recombinant OSGPR114 or OSGPR78

A broad variety of host cells can be used to study heterologouslyexpressed proteins. These cells include but are not limited to mammaliancell lines such as; Cos-7, CHO, LM(tk−), HEK293, RH7777, Jurkat, HCT4,RBL243, COS7 cells etc.; insect cell lines such as; Sf9, Sf21, etc.;amphibian cells such as Xenopus oocytes; assorted yeast strains;assorted bacterial cell strains; and others. Culture conditions for eachof these cell types is specific and is known to those familiar with theart.

COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/mL penicillin/100/g/mL streptomycin) at 37° C., 5%CO2. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days.

CHO cells are grown on 150 mm plates in HAM's F-12 medium withsupplements (10% bovine calf serum, 4 mM L-glutamine and 100 units/mLpenicillin/100 μg/mL streptomycin) at 37° C., 5% CO2. Stock plates ofCHO cells are trypsinized and split 1:8 every 3-4 days.

Transient Expression

DNA encoding proteins to be studied can be transiently expressed in avariety of mammalian, insect, amphibian, yeast, bacterial and other celllines by several methods, such as, calcium phosphate-mediated,DEAE-dextran mediated, liposomal-mediated, viral-mediated,electroporation-mediated and microinjection delivery. Each of thesemethods may require optimization of assorted experimental parametersdepending on the DNA, cell line, and the type of assay to besubsequently employed.

A typical protocol for the electroporation method as applied to Cos-7cells is described as follows. Cells to be used for transfection aresplit 24 hours prior to the transfection to provide flasks which aresubconfluent at the time of transfection. The cells are harvested bytrypsinization resuspended in their growth media and counted. 5×10⁶cells are suspended in 300 μL of DMEM and placed into an electroporationcuvette. 8 μg of receptor DNA plus 8 μg of any additional DNA needed(e.g. G protein expression vector, reporter construct, antibioticresistance marker, mock vector, etc.) is added to the cell suspension,the cuvette is placed into a BioRad Gene Pulser and subjected to anelectrical pulse (Gene Pulser settings: 0.25 kV voltage, 950 μFcapacitance). Following the pulse, 800 μL of complete DMEM is added toeach cuvette and the suspension transferred to a sterile tube. Completemedium is added to each tube to bring the final cell concentration to1×10⁵ cells/100 μL. The cells are then plated as needed depending uponthe type of assay to be performed.

Stable Expression

Heterologous DNA can be stably incorporated into host cells, causing thecell to perpetually express a foreign protein. Methods for the deliveryof the DNA into the cell are similar to those described above fortransient expression but require the co-transfection of an ancillarygene to confer drug resistance on the targeted host cell. The ensuingdrug resistance can be exploited to select and maintain cells that havetaken up the DNA. An assortment of resistance genes are availableincluding but not restricted to neomycin, kanamycin, and hygromycin. Forthe purposes of studies concerning the receptor of this invention,stable expression of a heterologous receptor protein is typicallycarried out in, mammalian cells including but not necessarily restrictedto, CHO, HEK293, LM(tk−), ASPC-1, RH7777, Jurkat, HCT4, RBL243, COS7cells etc.

In addition native cell lines that naturally carry and express thenucleic acid sequences for the receptor may be used without the need toengineer the receptor complement.

Quantitative Expression Analysis of OSGPR114 and OSGPR78

Quantitative RT-PCR by the ABI Prism 7700 Sequence Detector (TaqMan) wasused to determine tissue specific expression of OSGPR114/OSGPR78 in avariety of human tissues and cell lines. Initially, RNA is isolated fromthese tissues according to Clontech's Nucleospin RNA II Protocol and kitfor cultured cells. The cells are lysed using a kit buffer containingB-mercaptoethanol. The lysate is filtered and cleared using ethanol. TheRNA in solution binds to provided nucleospin columns. Once bound to thecolumn, the RNA is treated with a DNase, washed with buffers supplied inthe kit, and eluted with nuclease-free water in ribonuclease-free tubes.The RNA yield is estimated using UV spectrophotometry with a conversionof 1.0 A260 unit RNA=40 μg/mL.

1 μg RNA is used for first-strand cDNA synthesis with SUPERSCRIPT II(Invitrogen) in a final RT-PCR reaction volume of 20 μl. The initial 11μl reaction mixture containing 0.5 μg oligo (dT) 12-18, 1 μg RNA, and500 μM dNTP mix, was heated to 65° C. for 5 minutes followed by a briefcooling on ice. This reaction volume was increased to 19 μl using 1×first-strand buffer, 0.01M DTT, and 40 units RNaseOUT-RibonucleaseInhibitor (Ambion), and the reactions were heated to 42° C. for 2minutes. Finally, 200 units SUPERSCRIPT II were added to the (+)reactions and water was added to the (−) No RT control reactions, makingthe final RT-PCR reaction volume 20 μl. The reactions were cycled onceat 42° C. for 30 minutes, 45° C. for 15 minutes, 49° C. for 15 minutes,and 72° C. for 10 minutes. The total volume of the cDNA reaction was 20μl containing lug RNA starting material. This starting material wasdiluted 1:20 in the reaction mixture. Therefore, the theoretical cDNAyield was 50 ng/μl. Samples were diluted two-fold to titrate thetheoretical yield to 25 ng/μl.

cDNA (+) and No RT control samples were plated in 96 well PCR plates(Applied Biosystems) (with optical caps) for TaqMan/expression profilingusing the expression oligonucleotide listed. Amplification TaqMan mixincludes 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 0.9 μMprimer, 0.3 μM TaqMan probe, and 25 ng cDNA sample, for a total reactionvolume of 25 μl. The reactions are cycled 40 times at 50° C. for 2minutes, 95° C. for 10 minutes, 95° C. for 15 seconds, and 60° C. for 1minute. A control plate using TFIIB primers and probe was used toconfirm the presence of cDNA in the (+) samples, as well as, the absenceof cDNA in the No RT controls. TFIIB is a transcription factor expressedin most cell lines. Therefore, TFIIB data is used fornormalizing/standardizing expression data (Data are expressed as a ratioof gene expression/TFIIB expression).

The fluorogenic (TaqMan) probes (e.g. FIG. 12) are designed such thatthe oligonucleotide contains a 5′-reporter dye and a downstream,3′-quencher dye. The reporter dye, such as FAM (6-carboxy-fluorescein),is a fluorescent dye linked to the 5′ end of the nucleotide via acovalent bond. Located at the 3′ end, TAMRA(6-carboxy-tetramethyl-rhodamine) is responsible for quenching thefluorescent reporter dye. This suppressive activity is due to the closeproximity of the reporter dye and the quencher dye when the probe isintact. TaqMan probes are designed to hybridize to a sequence regioninternal to the target gene and no other gene (determined by BLASTanalysis of designed oligonucleotide sequences). Gene-specific forwardand reverse primers are also designed to hybridize the sequence regionsflanking the probe hybridization sequence.

TaqMan PCR master mix contains AmpliTaq Gold polymerase. Because 5′-3′nuclease activity is characteristic of Taq polymerases, thesepolymerases have the ability to cleave nucleotides off the template DNAstrand during 5′ to 3′ polymerization and amplification. Therefore, in aPCR reaction containing OSGPR114- or OSGPR78-Tqn1 in addition toOSGPR114 or OSGPR78 forward and reverse primers, the receptor probe willbe cleaved from the DNA template during OSGPR114/OSGPR78-F1 primerextension. Upon cleavage of the 5′ end of the probe, the reporter dye isreleased into solution and separated from the quencher dye. This resultsin the increase of reporter fluorescence during every PCR cycle.Therefore, the greater the fluorescence, the greater the amplification(and expression) of the target gene. The amount of fluorescence measuredby the ABI Prism 7700 Sequence Detector is related to the amount ofexpressed OSGPR114 or OSGPR78 in genomic DNA equivalents.

TaqMan expression profiling was completed for OSGPR114 and OSGPR78 usingMarathon-Ready cDNAs prepared from normal human tissues (Clontech), andin-house cDNAs made from tumor cell lines and from matched pairs oftumor and normal tissue using the aforementioned protocols.

Expression of OSGPR114 in Mammalian Cells

To set up secondary assays for the OSGPR114, cDNA encoding for theOSGPR114 (plasmid OP-T7403, pDNA3.1/OSGPR114, zeo) was stablytransfected into CHO cells. Cells were selected in growing mediumcontaining 0.2 mg/mL zeocin. Four stable clones were isolated by limiteddilution. Total RNAs were isolated from untransfected CHO cells as wellCHO/OSGPR114 clones. First strand cDNA was synthesized and used astemplates. mRNA level was quantitated by PCR with Taqman using theOSGPR114 specific primers and the probe.

Membrane Preparations

Cell membranes expressing the receptor protein according to thisinvention are useful for certain types of assays including but notrestricted to ligand binding assays, GTP-γ-S binding assays, and others.The specifics of preparing such cell membranes may in some cases bedetermined by the nature of the ensuing assay but typically involveharvesting whole cells and disrupting the cell pellet by sonication inice cold buffer (e.g. 20 mM Tris-HCl, 5 mM EDTA, pH 7.4). The resultingcrude cell lysate is cleared of cell debris by low speed centrifugationat 200×g for 5 min at 4° C. The cleared supernatant is then centrifugedat 40,000×g for 20 min at 4° C., and the resulting membrane pellet iswashed by suspending in ice cold buffer and repeating the high speedcentrifugation step. The final washed membrane pellet is resuspended inassay buffer. Protein concentrations are determined by the method ofBradford (1976) using bovine serum albumin as a standard. The membranesmay be used immediately or frozen for later use.

Alternatively, plasma membrane-containing P2 particulate fractions arereadily prepared from cell pastes frozen at −80° C. after harvest. Allprocedures are carried out at 4° C. Cell pellets are resuspended in 1 mLof 10 mM Tris-HCl and 0.1 mM EDTA, pH 7.5 (buffer A) and byhomogenisation for 20 s with a polytron homogeniser followed by passage(5 times) through a 25-guage needle. Cell lysates are centrifuged at1,000 g for 10 min in a microcentrifuge to pellet the nuclei andunbroken cells and P2 particulate fractions are recovered bymicrocentrifugation at 16,000 g for 30 min. P2 particulate fractions areresuspended in buffer A and stored at −80° C. until required. Proteinconcentrations are determined using the bicinchoninic acid (BCA)procedure (Smith, P. K. et al. (1985) Analytical Biochemistry,150:76-85) using BSA as a standard.

Generation of Baculovirus

The coding region of DNA encoding the OSGPR114 or OSGPR78 receptordisclosed herein may be subcloned into pBlueBacIII into existingrestriction sites or sites engineered into sequences 5′ and 3′ to thecoding region of the polypeptides. To generate baculovirus, 0.5 μg ofviral DNA (BaculoGold) and 3 μg of DNA construct encoding a polypeptidemay be co-transfected into 2×10⁶ Spodoptera frugiperda insect Sf9 cellsby the calcium phosphate co-precipitation method, as outlined byPharmingen (in “Baculovirus Expression Vector System: Procedures andMethods Manual”). The cells then are incubated for 5 days at 27° C.

The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

Yeast Assays

The yeast cell-based reporter assays have previously been described inthe literature (e.g. see Miret J. J., et. al. (2002) J. Biol. Chem.277:6881-6887; Campbell R. M., et. al. (1999) Bioorg Med. Chem. Lett.9:2413-2418; King K., et. al. (1990) Science. 250:121-123); WO 99/14344;WO 00/12704; and U.S. Pat. No. 6,100,042). Briefly, yeast cells havebeen engineered such that the endogenous yeast G-alpha (GPA1) has beendeleted and replaced with G-protein chimeras constructed using multipletechniques. Additionally the endogenous yeast alpha-cell GPCR, STE3 hasbeen deleted to allow for a homologous expression of a mammalian GPCR ofchoice. In the yeast, elements of the pheromone signaling transductionpathway, which are conserved in eukaryotic cells (for example, themitogen-activated protein kinase pathway), drive the expression of Fus1.By placing β-galactosidase (LacZ) under the control of the Fus1 promoter(Fus1p), a system has been developed whereby receptor activation leadsto an enzymatic read-out. Different yeast strains (e.g. FIG. 6) denotethe presence of different G-alpha chimeras (see “yeast reporter assays”in Results section below for definitions).

Yeast cells are transformed by an adaptation of the Lithium acetatemethod described by Agatep et. al. (Agatep, R., et. al. (1998)Transformation of Saccharomyces cerevisiae by the lithiumacetate/single-stranded carrier DNA/polyethylene glycol(LiAc/ss-DNA/PEG) protocol. Technical Tips Online, Trends Journals,Elsevier). Briefly, yeast cells are grown overnight on yeast tryptoneplates (YT). Carrier single-stranded DNA (10 μg), 2 μg of each of twoFus1p-LacZ reporter plasmids (one with URA3 selection marker and onewith TRP1), 2 μg of OSGPR114 or OSGPR78 in yeast expression vector (2μorigin of replication) and a lithium acetate/polyethylene glycol/TEbuffer is pipetted into an Epindorf tube. The yeast expression plasmidcontaining the receptor/no receptor control has a LEU2 marker. Yeastcells are inoculated into this mixture and the reaction proceeds at 30°C. for 60 minutes. The yeast cells are then heat-shocked at 42° C. for15 minutes. The cells are then washed and spread on selection plates.The selection plates are synthetic defined yeast media minus LEU, URAand TRP (SD-LUT). After incubating at 30° C. for 2-3 days, colonies thatgrow on the selection plates are then tested in the LacZ assay.

In order to perform fluorometric enzyme assays for β-galactosidase,yeast cells were grown overnight in liquid SD-LUT media to anunsaturated concentration (i.e. the cells are still dividing and havenot yet reached stationary phase). They are diluted in fresh media to anoptimal assay concentration and 90 μL of yeast cells were added to96-well black polystyrene plates (Costar). Compounds, dissolved inethanol and diluted in a 1% BSA solution to 10× concentration, wereadded to the plates and the yeast are placed at 30° C. for 4 h. After 4h, the substrate for the β-galactosidase is added to each well. Thesubstrate may yield a fluorescent or colorimetric read-out upon theactivity of the β-galactosidase. In these experiments, Fluorescein di(β-D-galactopyranoside) was used (FDG), a substrate for the enzyme thatreleases fluorescein, allowing a fluorometric read-out. 20/L of 500 μMFDG was used. After incubation of the cells with the substrate for 30-60mins, 20 μL of 1 M sodium carbonate is added to terminate the reactionand enhance the fluorescent signal. The plates were then read in afluorometer at 485/535 nm. As an alternate read-out system, the yeastare also engineered with a Fus1p-HIS gene which means that activation ofthe receptor can also be measured through the growth of the yeast in ahistamine deficient media.

Melanophore Assays

Polypeptide of the invention can be heterologously expressed in Xenopuslaevis melanophores and its activation can be measured by eithermelanosome dispersion or aggregation. Basically, melanosome dispersionis promoted by activation of adenylate cyclase or phospholipase C i.e.Gs and Gq mediated signalling, respectively, whereas aggregation resultsfrom activation of Gi/o G proteins resulting in inhibition of adenylatecyclase. Hence, ligand activation of the OSGPR114 or OSGPR78 can bemeasured simply by measuring the change in light transmittance throughthe cells or by imaging the cell response.

Assays for Compound Screening

OSGPR114 or OSGPR78 modulator activity can be determined by contactingcells expressing an OSGPR114 or OSGPR78 polypeptide of the inventionwith a substance under investigation and by monitoring the effectmediated by the polypeptides. The cells expressing the polypeptide maybe in vitro or in vivo. The polypeptide of the invention may benaturally or recombinantly expressed. Preferably, the assay is carriedout in vitro using cells expressing recombinant polypeptide. Typically,receptor activity can be monitored indirectly by measuring aGi/o-coupled readout. Gi/o coupled readout can typically be monitoredusing an electrophysiological method to determine the activity ofG-protein regulated Ca⁺ or K⁺ channels or by using a fluorescent dye tomeasure changes in intracellular Ca²⁺ levels. Other methods that cantypically be used to monitor receptor activity involve measuring levelsof or activity of labeled bound GTPγs or cAMP.

Preferably, control experiments are carried out on cells which do notexpress the polypeptide of the invention to establish whether theobserved responses are the result of activation of the polypeptide.

Mammalian cells, such as HEK293, CHO, COS7, RH7777, Jurkat, HCT4, andRBL243 cells over-expressing the protein of choice are generated for usein the assay. Cell lines which maybe employed as suitable hosts includei) CHO cells transfected to stably express PLC 2, a PLC isoform whichallows Gi/o G proteins to elicit Ca²⁺ mobilization, or ii) CHO cellstransfected to stably express the Gq family G-protein G₁₆ together witha suitable reporter construct gene e.g. the Gq responsive NFAT (nuclearfactor activator of T cells) promoter controlling expression ofluciferase. Expression of G₁₆ permits a wide variety of non-Gq coupledreceptors to mobilize Ca²⁺.

96 and 384 well plate high throughput screens (HTS) are employed usinga) fluorescence based calcium indicator molecules, including but notlimited to dyes such as Fura-2, Fura-Red, Fluo 3 and Fluo 4 (MolecularProbes); or b) reporter gene read-out. Secondary screening involves thesame technology. A brief screening assay protocol is as follows:—

Mammalian cells stably over-expressing the protein are cultured in blackwall, clear bottom, tissue culture coated 96 or 384 well plates with avolume of 100 μL cell culture medium in each well 3 days before use in aFLIPR (Fluorescence Imaging Plate Reader—Molecular Devices). Cells wereincubated with 4 μM Fluo-3 at 30° C. in 5% CO₂ for 90 mins and thenwashed once in Tyrodes buffer containing 3 mM probenecid. Basalfluorescence is determined prior to compound additions. Activationresults in an increase in intracellular calcium which can be measureddirectly in the FLIPR.

The binding of a modulator to a polypeptide of the invention can also bedetermined directly. For example, a radiolabeled test substance can beincubated with the polypeptide of the invention and binding of the testsubstance to the polypeptide can be monitored. Typically, theradiolabeled test substance can be incubated with cell membranescontaining the polypeptide until equilibrium is reached. The membranescan then be separated from a non-bound test substance and dissolved inscintillation fluid to allow the radioactive content to be determined byscintillation counting. Non-specific binding of the test substance mayalso be determined by repeating the experiment in the presence of asaturating concentration of a non-radioactive ligand.

Labeled Ligand Binding Assays

Cells expressing the receptor according to this invention may be used toscreen for ligands for said receptors, for example, by labeled ligandbinding assays. Once a ligand is identified the same assays may be usedto identify agonists or antagonists of the receptor that may be employedfor a variety of therapeutic purposes.

In an embodiment, labeled ligands are placed in contact with eithermembrane preparations or intact cells expressing the receptor inmulti-well microtiter plates, together with unlabeled compounds, andbinding buffer. Binding reaction mixtures are incubated for times andtemperatures determined to be optimal in separate equilibrium bindingassays. The reaction is stopped by filtration through GF/B filters,using a cell harvester, or by directly measuring the bound ligand. Ifthe ligand was labeled with a radioactive isotope such as ³H, ¹⁴C, ¹²⁵I,³⁵S, ³²P, ³³P, etc., the bound ligand may be detected by using liquidscintillation counting, scintillation proximity, or any other method ofdetection for radioactive isotopes. For example, see above for ³H-LPAassay. If the ligand was labeled with a fluorescent compound, the boundlabeled ligand may be measured by methods such as, but not restrictedto, fluorescence intensity, time resolved fluorescence, fluorescencepolarization, fluorescence transfer, or fluorescence correlationspectroscopy. In this manner agonist or antagonist compounds that bindto the receptor may be identified as they inhibit the binding of thelabeled ligand to the membrane protein or intact cells expressing thereceptor. Non-specific binding is defined as the amount of labeledligand remaining after incubation of membrane protein in the presence ofa high concentration (e.g., 100-1000×K_(D)) of unlabeled ligand. Inequilibrium saturation binding assays membrane preparations or intactcells transfected with the receptor are incubated in the presence ofincreasing concentrations of the labeled compound to determine thebinding affinity of the labeled ligand. The binding affinities ofunlabeled compounds may be determined in equilibrium competition bindingassays, using a fixed concentration of labeled compound in the presenceof varying concentrations of the displacing ligands.

For example, in one method employed herein, membranes from untransfectedCHO cells as well as OSGPR114 stable clones were prepared using themethod described by Griffin et al (1998). Membranes were homogenized inbuffer containing 50 mM Tris, 5 mM MgCl₂, 2.5 mM EDTA, 0.1% fatty acidfree BSA, pH 7.4. Assays were performed in a reaction mixture containing501 of membrane suspension (˜3 g/well), 25 μl of [³H]-LPA (finalconcentration=56 nM:25 μl aliquot counted to determine preciseconcentration employed). Nonspecific binding was measured in thepresence of 10 μM LPA. Incubations were carried out in triplicate for 60minutes at 37° C. Reactions were terminated by rapid filtration overGF/B filters presoaked in ice cold wash buffer for 90 minutes. Thefilters were then washed 6 times with 0.3 mL of ice cold 50 mM Tris, 5mM MgCl₂, 2.5 mM EDTA, 0.1% fatty acid free BSA, pH 7.4. The filterswere dried, covered with Microscint-20, dark adapted for 5 minutes, andcounted on a 2 minute tritium protocol on a Packard TopCount LSC.

Functional Assays

Cells expressing the OSGPR114 or OSGPR78 receptor DNA may be used toscreen for additional ligands to the OSGPR114 or OSGPR78 receptor usingfunctional assays. The same assays, in the presence of a ligand, may beused to identify agonists or antagonists of the OSGPR114 or OSGPR78receptor that may be employed for a variety of therapeutic purposes. Itis well known to those in the art that the over-expression of a GPCR canresult in the constitutive activation of intracellular signalingpathways. In the same manner, over-expression of the OSGPR114 or OSGPR78receptor in any cell line as described above, can result in theactivation of the functional responses described below, and any of theassays herein described can be used to screen for both agonist andantagonist ligands of the OSGPR114 or OSGPR78 receptor.

A wide spectrum of assays can be employed to screen for the presence ofOSGPR114 or OSGPR78 receptor ligands. These assays range fromtraditional measurements of total inositol phosphate accumulation, cAMPlevels, intracellular calcium mobilization, and potassium or sodiumcurrents, for example; to systems measuring these same second messengersbut which have been modified or adapted to be of higher throughput, moregeneric and more sensitive; to cell based assays reporting more generalcellular events resulting from receptor activation such as metabolicchanges, differentiation, cell division/proliferation. Description ofseveral such assays follow.

Adenylate Cyclase/Cyclic AMP (cAMP) Assay

The adenylate cyclase assay is performed with an alpha-screen cAMP assaykit (Promega). The manufacturer's protocol is followed. Briefly, cellsknown to endogenously express moderate levels of OSGPR114 or OSGPR78 arelysed from the culture flask using a lysis buffer (0.1% BSA in PBScontaining 20 mM rolipram and 0.54% TWEEN20), washed with PBS anddiluted in assay buffer (0.1% BSA in PBS containing 20 mM rolipram) to aconcentration of 600,000 cells/mL. White polystyrene 96-well plates areused for the assay. The following reagents are added to the wells: 5 mLforskolin (4× in assay buffer, 4×10⁻⁴ M) or 5 μl assay buffer+5 mLLPA/vehicle control (LPA (4×) is dissolved in a 0.1% BSA in watersolution)+5 mL acceptor beads (Stock solution is 90 mg/mL; 12 mL/0.5 mLstimulation buffer is used for the assay)+5 mL cells to initiate assay.The plate is incubated at room temperature for 30 min. For the standardcAMP curve used to quantify the data, 5 mL water+5 mL 4× cAMP in assaybuffer+5 mL acceptor beads+5 mL cells are added to the plate. Toterminate the assay, 10 μl of the donor beads solution is added (30 nMbiotin-cAMP/60 mg streptavidin-donor beads in 1 mL lysis buffer). Theplates are incubated overnight, protected from light. A Fusion-aHTcounter was used to read the plates according to the kit's instructions.

In an alternative assay for measuring camp levels, a Cre-SEAP assay isused. HEK-293-Galpha16 cells stably expressing the cre-SEAP (cyclicAMPresponse element containing promoter driving expression of the secretedalkaline phosphatase gene) reporter construct, and transientlytransfected with either the OSGPR114 or OSGPR78 vector or empty vectorcontrol, are grown to between ˜0.5×10⁶ and 1×10⁷ cells/dish and seededat 4×10⁴ cells/well on poly-D-lysine coated clear-bottom 96-well tissueculture plates. The cells are left to adhere to the plates in media for24 h at 37° C. The media is aspirated and the cells arrested by serumstarvation for a further 24 h in 200 mL of colorless DMEM.

After serum arrest, the starvation media is aspirated and 90 mL freshcolorless DMEM containing 0.1% BSA is added. 10 μL of 10× compound (orDMSO/H2O control) is added and the plates incubated at 37° C. for 7 h.Conditioned media (12.5 mL) is then transferred to a 96 well whiteflat-bottom plate. Next, 37.5 mL of 1× Tropix Dilution Buffer is addedto the transferred culture media. 50 mL of Tropix Assay Buffer is alsoadded. The plate is then incubated at room temperature for 5 minutes.Finally, 50 mL Tropix Reaction Buffer Diluent containing a 1:20 dilutionof the CSPD Chemiluminescent Substrate is added. The plate is incubatedat room temperature for 60 minutes and luminescence measured.

The receptor-mediated stimulation or inhibition of cyclic AMP (cAMP)formation by adenylate cyclase may also be assayed in cells expressingthe receptors by the following assay. According to this method, cellsare plated in 96-well plates or other vessels and preincubated in abuffer such as HEPES buffered saline (NaCl (150 mM), CaCl₂ (1 mM), KCl(5 mM), glucose (10 mM)) supplemented with a phosphodiesterase inhibitorsuch as 5 mM theophylline, with or without protease inhibitor cocktail(For example, a typical inhibitor cocktail contains 2 μg/mL aprotinin,0.5 mg/mL leupeptin, and 10 μg/mL phosphoramidon.) for 20 min at 37° C.,in 5% CO₂. Test compounds are added with or without 10 mM forskolin andincubated for an additional 10 min at 37° C. The medium is thenaspirated and the reaction stopped by the addition of 100 mM HCl orother methods. The plates are stored at 4° C. for 15 min, and the cAMPcontent in the stopping solution is measured by radioimmunoassay.Radioactivity may be quantified using a gamma counter equipped with datareduction software. Specific modifications may be performed to optimizethe assay for the receptor or to alter the detection method of cAMP.

According to a further method, cells are washed 2 times with HEPESbuffered saline, as described above, and incubated overnight. On the dayof the experiment, cells are washed 2 times with HEPES supplemented witha phosphodiesterase inhibitor such as 5 mM theophylline, with or withoutprotease inhibitor cocktail (For example, a typical inhibitor cocktailcontains 21 g/mL aprotinin, 0.5 mg/mL leupeptin, and 10 μg/mLphosphoramidon.) for 20 min at 37° C., in 5% CO₂. Test compounds areadded with or without 10 mM forskolin and incubated for an additional 10min at 37° C. The medium is then aspirated and the reaction stopped bythe addition of 100 mM HCl or other methods. The plates are stored at 4°C. for 15 min, and the cAMP content in the stopping solution is measuredby radioimmunoassay. Radioactivity may be quantified using a gammacounter equipped with data reduction software. Specific modificationsmay be performed to optimize the assay for the receptor or to alter thedetection method of cAMP.

Arachidonic Acid Release Assay

Cells expressing the receptor are seeded into 96 well plates or othervessels and grown for 3 days in medium with supplements ³H-arachidonicacid (specific activity=0.75 μCi/mL) is delivered as a 100 μL aliquot toeach well and samples are incubated at 37° C., 5% CO₂ for 18 hours. Thelabeled cells are washed three times with medium. The wells are thenfilled with medium and the assay is initiated with the addition of testcompounds or buffer in a total volume of 250 μL. Cells are incubated for30 min at 37° C., 5% CO₂. Supernatants are transferred to a microtiterplate and evaporated to dryness at 75° C. in a vacuum oven. Samples arethen dissolved and resuspended in 25 μL distilled water. Scintillant(300 μL) is added to each well and samples are counted for ³H in aTrilux plate reader. Data are analyzed using nonlinear regression andstatistical techniques available in the GraphPAD Prism package (SanDiego, Calif.).

Inositol Phosphate Assay

OSGPR114 or OSGPR78 receptor-mediated activation of the inositolphosphate (IP) second messenger pathways can be assessed by radiometricmeasurement of IP products.

In a 96 well microplate format assay, cells are plated at a density of70,000 cells per well and allowed to incubate for 24 hours. The cellsare then labeled with 0.5 μCi [³H]-myo-inositol overnight at 37° C., 5%CO₂. Immediately before the assay, the medium is removed and replacedwith 90 μL of PBS containing 10 mM LiCl. The plates are then incubatedfor 15 min at 37° C., 5% CO₂. Following the incubation, the cells arechallenged with agonist (10 μL/well; 10× concentration) for 30 min at37° C., 5% CO₂. The challenge is terminated by the addition of 100 μL of50% v/v trichloroacetic acid, followed by incubation at 4° C. forgreater than 30 minutes. Total IPs are isolated from the lysate by ionexchange chromatography. Briefly, the lysed contents of the wells aretransferred to a Multiscreen HV filter plate (Millipore) containingDowex AG1-X8 (200-400 mesh, formate form). The filter plates areprepared adding 100 μL of Dowex AG1-X8 suspension (50% v/v, water:resin) to each well. The filter plates are placed on a vacuum manifoldto wash or elute the resin bed. Each well is first washed 2 times with200 μL of 5 mM myo-inositol. Total [³H]inositol phosphates are elutedwith 75 μL of 1.2M ammonium formate/0.1M formic acid solution into96-well plates. 200 μL of scintillation cocktail is added to each well,and the radioactivity is determined by liquid scintillation counting.

Intracellular Calcium Mobilization Assays

The intracellular free calcium concentration may be measured bymicrospectrofluorimetry using the fluorescent indicator dye Fura-2/AM(Bush et. al., (1991) J. Neurochem. 57: 562-574). Cells expressing thereceptor are seeded onto a 35 mm culture dish containing a glasscoverslip insert and allowed to adhere overnight. Cells are then washedwith HBS and loaded with 100 μL of Fura-2/AM (10 μM) for 20 to 40 min.After washing with HBS to remove the Fura-2/AM solution, cells areequilibrated in HBS for 10 to 20 min. Cells are then visualized underthe 40× objective of a Leitz Fluovert FS microscope and fluorescenceemission is determined at 510 nM with excitation wavelengths alternatingbetween 340 nM and 380 nM. Raw fluorescence data are converted tocalcium concentrations using standard calcium concentration curves andsoftware analysis techniques.

In another method, the measurement of intracellular calcium can also beperformed on a 96-well (or higher) format and with alternativecalcium-sensitive indicators, preferred examples of these are: aequorin,Fluo-3, Fluo-4, Fluo-5, Calcium Green-1, Oregon Green, and 488 BAPTA.After activation of the receptors with agonist ligands the emissionelicited by the change of intracellular calcium concentration can bemeasured by a luminometer, or a fluorescence imager; a preferred exampleof this is the fluorescence imager plate reader (FLIPR).

Cells expressing the receptor of interest are plated into clear,flat-bottom, black-wall 96-well plates (Costar) at a density of80,000-150,000 cells per well and allowed to incubate for 48 hr at 5%CO₂, 37° C. The growth medium is aspirated and 100 μL of loading mediumcontaining fluo-3 dye is added to each well. The loading mediumcontains: Hank's BSS (without phenol red) (Gibco), 20 mM HEPES (Sigma),0.1 or 1% BSA (Sigma), dye/pluronic acid mixture (e.g. 1 mM Flou-3, AM(Molecular Probes) and 10% pluronic acid (Molecular Probes) mixedimmediately before use), and 2.5 mM probenecid (Sigma) (prepared fresh).The cells are allowed to incubate for about 1 hour at 5% CO₂, 37° C.

During the dye loading incubation the compound plate is prepared. Thecompounds are diluted in wash buffer (Hank's BSS (without phenol red),20 mM HEPES, 2.5 mM probenecid) to a 4× final concentration andaliquoted into a clear v-bottom plate (Nunc). Following the incubationthe cells are washed to remove the excess dye. A Denley plate washer isused to gently wash the cells 4 times and leave a 100 μL final volume ofwash buffer in each well. The cell plate is placed in the center trayand the compound plate is placed in the right tray of the FLIPR. TheFLIPR software is setup for the experiment, the experiment is run andthe data are collected. The data are then analyzed using an excelspreadsheet program. Antagonist ligands are identified by the inhibitionof the signal elicited by agonist ligands.

For example, in one method employed herein, CHO and CHO-114 cells wereseeded at a concentration of 50K cells/well in 200 mL growth media inclear, flat-bottom 96-well black assay plates and incubated overnight at37 C. The growth media is aspirated from the cells and a dye solution(100 mL) added to each well. The dye solution consists of 2 mMprobenecid, 2 mM HBSS, 10 mM Fluo-3 in a growth media minus antibioticssolution. The cells are loaded with the dye for 1 h at 37 C beforeaspiration and rinsing with wash buffer (20 mM HBSS+2 mM Probenecid).Compound plates are then prepared with 4× compound/control. 150 ul ofwash buffer is added to each well. Once the FLIPR is fully calibrated,compound is added to the plate and the release of calcium measured using0.4 exposure length and a laser set at 0.3 W.

GTPγS Functional Assay

Membranes from cells expressing the receptor are suspended in assaybuffer (e.g., 50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, 10 μM GDP, pH 7.4)with or without protease inhibitors (e.g., 0.1% bacitracin). Membranesare incubated on ice for 20 minutes, transferred to a 96-well Milliporemicrotiter GF/C filter plate and mixed with GTP.γ⁵S (e.g., 250,000cpm/sample, specific activity .about.1000 Ci/mmol) plus or minusunlabeled GTPγS (final concentration=100 μM). Final membrane proteinconcentration.apprxeq.90 μg/mL. Samples are incubated in the presence orabsence of test compounds for 30 min. at room temperature, then filteredon a Millipore vacuum manifold and washed three times with cold (4° C.)assay buffer. Samples collected in the filter plate are treated withscintillant and counted for ³⁵S in a Trilux (Wallac) liquidscintillation counter. It is expected that optimal results are obtainedwhen the receptor membrane preparation is derived from an appropriatelyengineered heterologous expression system, i.e., an expression systemresulting in high levels of expression of the receptor and/or expressingG-proteins having high turnover rates (for the exchange of GDP for GTP).GTPγS assays are well-known to those skilled in the art, and it iscontemplated that variations on the method described above, such as aredescribed by Tian et al. (1994) Molecular Pharm. 45: 524-553 or Lazarenoand Birdsall (1993) Br. J. Pharmacol. 109: 1120-1127, may be used.

Alternatively, high affinity [³⁵S]-GTPγS binding assays are performed in96-well format using a method modified from Wieland and Jakobs (Wieland,T. & Jakobs, K. H. (1994) Method. Enzymol. 237: 3-13). Membranes (10 μgper point) are diluted to about 0.1 mg/mL in assay buffer (20 mM HEPES,100 mM NaCl, 10 mM MgCl₂, pH7.4) supplemented with saponin (10 mg/l) andpre-incubated with 40 μM GDP. Various concentrations of ligand (e.g.LPA) are added, followed by [³⁵S]-GTPγS (˜1200 Ci/mmol, Amersham) at 0.3nM (total vol. of 100 [d) and binding is allowed to proceed at roomtemperature for 30 min. Non-specific binding is determined by theinclusion of 0.6 mM GTP. Wheatgerm agglutinin SPA beads (Amersham; 0.5mg) in 25/L assay buffer are added and the whole is incubated at roomtemperature for 30 min with agitation. Plates are centrifuged at 1500 gfor 5 min and bound [³⁵S]-GTPγS is determined by scintillation counting.

Alternatively, instead of [³⁵S]-GTPγS, a time-resolved fluorescencebased GTP binding assay using a non-radioactive, non-hydrolyzable GTPanalog such as GTP-Eu (Perkin Elmer™) can be employed.

Furthermore, several methods that increase the utility of such assaysoutside of the pertussis-toxin-sensitive G-proteins can be employed.Methods involving the expression of GPCRs, and also G proteins ofinterest, in various insect cell lines that express low levels ofG-protein orthologues can be used (e.g. see Windh, R. T. and Manning, D.R. (2002) Methods Enzymol. 344:3-14). Introduction of a GPCR and amammalian G protein into such cells using baculoviral-based expressionvectors can provide high signal to background in [³⁵S]-GTPγS bindingassays. Immunocapture of [³⁵S]-GTPγS-bound G-proteins and GPCR-G-proteinfusion proteins can also be employed to enhance signal to background(e.g. see Milligan, G. (2003) Trends in Pharmacol. Sci. 24:87-90).

For example, in one method employed herein, membranes from untransfectedCHO cells as well as OSGPR114 stable clones were prepared using themethod described by Griffin et al (1998). Membranes were homogenized inbuffer containing 50 mM Tris, 3 mM MgCl2, 100 mM NaCl, 1 mM EDTA, 1 uMGDP, 0.1% fatty acid free BSA, 0.01% Saponin, pH 7.4. Assays werecarried out in a reaction mixture containing 50 mL of membranesuspension (˜5 mg/well), 50 mL of [³⁵S]-GTPgS binding (finalconcentration ˜0.5 nM: 50 mL aliquot counted to determine preciseconcentration employed) and either 50 mL of assay buffer, 50 mL ofunlabeled GTPgS (defining nonspecific binding: final concentration is 10mM) or 50 mL of each concentration of LPA. Incubations were carried outin triplicate for 60 minutes at 37° C. Reactions were terminated byrapid filtration over GF/B filters presoaked in ice cold wash buffer for90 minutes. The filters were then washed 6 times with 0.3 mL of ice coldwash buffer (50 mM Tris, 5 mM MgCl2, pH 7.4). The filters were dried,covered with Microscint-20, dark adapted for 5 minutes, and counted on a2 minute [35S] protocol on a Packard TopCount LSC.

Microphysiometric Assay

Because cellular metabolism is intricately involved in a broad range ofcellular events (including receptor activation of multiple messengerpathways), the use of microphysiometric measurements of cell metabolismcan in principle provide a generic assay of cellular activity arisingfrom the activation of any receptor regardless of the specifics of thereceptor's signaling pathway (Williams, C. (2000) Current OpinionBiotech. 11:42-46).

General guidelines for transient receptor expression, cell preparationand microphysiometric recording are described elsewhere (Salon, J. A.and Owicki, J. A., (1996) Meth. Neurosci. 25: 201-224). Typically cellsexpressing receptors are harvested and seeded at 3×10⁵ cells permicrophysiometer capsule in complete media 24 hours prior to anexperiment. The media is replaced with serum free media 16 hours priorto recording to minimize non-specific metabolic stimulation by assortedand ill-defined serum factors. On the day of the experiment the cellcapsules are transferred to the microphysiometer and allowed toequilibrate in recording media (low buffer RPMI 1640, no bicarbonate, noserum (Molecular Devices Corporation, Sunnyvale, Calif.) containing 0.1%fatty acid free BSA), during which a baseline measurement of basalmetabolic activity is established.

A standard recording protocol specifies a 100 μL/min flow rate, with a 2min total pump cycle which includes a 30 sec flow interruption duringwhich the acidification rate measurement is taken. Ligand challengesinvolve a 1 min 20 sec exposure to the sample just prior to the firstpost challenge rate measurement being taken, followed by two additionalpump cycles for a total of 5 min 20 sec sample exposure. Typically,drugs in a primary screen are presented to the cells at 10 μM finalconcentration. Follow up experiments to examine dose-dependency ofactive compounds are then done by sequentially challenging the cellswith a drug concentration range that exceeds the amount needed togenerate responses ranging from threshold to maximal levels. Ligandsamples are then washed out and the acidification rates reported areexpressed as a percentage increase of the peak response over thebaseline rate observed just prior to challenge.

MAP Kinase Assay

MAP kinase (mitogen activated kinase) may be monitored to evaluatereceptor activation. MAP kinase is activated by multiple pathways in thecell. A primary mode of activation involves the ras/raf/MEK/MAP kinasepathway. Growth factor (tyrosine kinase) receptors feed into thispathway via SHC/Grb-2/SOS/ras. Gi coupled receptors are also known toactivate ras and subsequently produce an activation of MAP kinase.Receptors that activate phospholipase C (such as Gq/G11-coupled) producediacylglycerol (DAG) as a consequence of phosphatidyl inositolhydrolysis. DAG activates protein kinase C which in turn phosphorylatesMAP kinase.

MAP kinase activation can be detected by several approaches. Oneapproach is based on an evaluation of the phosphorylation state, eitherunphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the test compound and then extracted with Laemmlibuffer. The soluble fraction is applied to an SDS-PAGE gel and proteinsare transferred electrophoretically to nitrocellulose or Immobilon.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

Another approach is based on evaluation of the MAP kinase activity via aphosphorylation assay. Cells are stimulated with the test compound and asoluble extract is prepared. The extract is incubated at 30° C. for 10min with γ-³²P-ATP, an ATP regenerating system, and a specific substratefor MAP kinase such as phosphorylated heat and acid stable proteinregulated by insulin, or PHAS-I. The reaction is terminated by theaddition of H₃PO₄ and samples are transferred to ice. An aliquot isspotted onto Whatman P81 chromatography paper, which retains thephosphorylated protein. The chromatography paper is washed and countedfor ³²P in a liquid scintillation counter. Alternatively, the cellextract is incubated with γ³² P ATP, an ATP regenerating system, andbiotinylated myelin basic protein bound by streptavidin to a filtersupport. The myelin basic protein is a substrate for activated MAPkinase. The phosphorylation reaction is carried out for 10 min at 30° C.The extract can then by aspirated through the filter, which retains thephosphorylated myelin basic protein. The filter is washed and countedfor ³²P by liquid scintillation counting.

Cell Proliferation Assay

Receptor activation of the receptor may lead to a mitogenic orproliferative response which can be monitored via ³H-thymidine uptake.When cultured cells are incubated with ³H-thymidine, the thymidinetranslocates into the nuclei where it is-phosphorylated to thymidinetriphosphate. The nucleotide triphosphate is then incorporated into thecellular DNA at a rate that is proportional to the rate of cell growth.Typically, cells are grown in culture for 1-3 days. Cells are forcedinto quiescence by the removal of serum for 24 hrs. A mitogenic agent isthen added to the media. Twenty-four hours later, the cells areincubated with ³H-thymidine at specific activities ranging from 1 to 10μCi/mL for 2-6 hrs. Harvesting procedures may involve trypsinization andtrapping of cells by filtration over GF/C filters with or without aprior incubation in TCA to extract soluble thymidine. The filters areprocessed with scintillant and counted for ³H by liquid scintillationcounting. Alternatively, adherent cells are fixed in MeOH or TCA, washedin water, and solubilized in 0.05% deoxycholate/0.1 N NaOH. The solubleextract is transferred to scintillation vials and counted for ³H byliquid scintillation counting.

Alternatively, cell proliferation can be assayed by measuring theexpression of an endogenous or heterologous gene product, expressed bythe cell line used to transfect the receptor, which can be detected bymethods such as, but not limited to, florescence intensity, enzymaticactivity, immunoreactivity, DNA hybridization, polymerase chainreaction, etc.

Assays for Chemotactic Cell Motility and Chemoinvasion

Chemoinvasion is measured by the ability of tumor cells to migratethrough a thick layer of MATRI-GEL during a prolonged incubation period(70 hours). This property is distinct from chemotactic cell motility,which involves a much shorter incubation period (20 hours) and a thinlayer of MATRI-GEL.

Assays are performed using transwell plates with polycarbonate membranefilters (pore size 8 μm) (Costar Scientific, Cambridge, Mass.). 50 μLaliguots of an aqueous solution of MATRI-GEL (Collaborative Research,Bedford, Mass.) containing 20 μg/mL (for chemotactic motility assay) or200 μg/mL (for chemoinvasion assay) are added to each well and driedovernight. The filter is fitted onto the lower chamber plate. The lowerchamber contains 0.6 mL medium containing a moltility factor, with orwithout the compound with activity on OSGPR114 or OSGPR78. To the upperchamber is added 100 μL of cell suspension (5×10⁴ cells/mL for invasionassay, 5×10⁵ cells/mL for motility assay), which is then incubated in 5%CO₂ at 37° C. for 70-72 hours (invasion assay) or 20 hours (motilityassay). After incubation, cells remaining in the upper chamber are wipedoff with a cotton swab, and cells which had migrated to the lowerchamber side of the filter are fixed in methanol for 30 seconds andstained with 0.05% toluidine blue. The filter is removed, the stain wassolubilized in 10% acetic acid (0.1 mL for invasion assay, 0.5 mL formotility assay), and color intensity (optical density) is quantitated byELISA reading at 630 nm. A linear relationship is observed between cellnumber and toluidine blue optical density.

Promiscuous Second Messenger Assays

It is not possible to predict, a priori and based solely upon the GPCRsequence, which of the cell's many different signaling pathways anygiven receptor will naturally use. It is possible, however, to coaxreceptors of different functional classes to signal through apre-selected pathway through the use of promiscuous G-alpha. subunits.For example, by providing a cell based receptor assay system with anendogenously supplied promiscuous G-alpha subunit such as G-alpha₁₅ orG-alpha₁₆ (i.e. G₁₅ or G₁₆) or a chimeric G-alpha subunit such asG-alpha_(qz), a GPCR, which might normally prefer to couple through aspecific signaling pathway (e.g., G_(s), G_(i), G_(q), G_(o), etc.), canbe made to couple through the pathway defined by the promiscuous Galphasubunit and upon agonist activation produce the second messengerassociated with that subunit's pathway. In the case of G-alpha₁₅,G-alpha₁₆ and/or G-alpha_(qz) this would involve activation of the G_(q)pathway and production of the second messenger IP₃. Through the use ofsimilar strategies and tools, it is possible to bias receptor signalingthrough pathways producing other second messengers such as Ca²⁺, cAMP,and K⁺ currents, for example (Milligan, G. and Rees, S. (1999) TIPS20:118-124).

It follows that the promiscuous interaction of the exogenously suppliedG-alpha subunit with the receptor alleviates the need to carry out adifferent assay for each possible signaling pathway and increases thechances of detecting a functional signal upon receptor activation.

Methods for Recording Currents in Xenopus Oocytes

Oocytes are harvested from Xenopus laevis and injected with mRNAtranscripts as previously described (Quick, M. W. and Lester, H. A.,(1994) Meth. Neurosci. 19: 261-279; Smith et. al. (1997) J. Biol. Chem.272: 24612-24616). The test receptor of this invention and G-alpha.subunit RNA transcripts are synthesized using the T7 polymerase(“Message Machine,” Ambion) from linearized plasmids or PCR productscontaining the complete coding region of the genes. Oocytes are injectedwith 10 ng of synthetic receptor RNA and incubated for 3-8 days at 17degrees. Three to eight hours prior to recording, oocytes are injectedwith 500 pg promiscuous G-alpha subunits mRNA in order to observecoupling to Ca²⁺ activated Cl-currents. Dual electrode voltage clamp(Axon Instruments Inc.) is performed using 3 M KCl-filled glassmicroelectrodes having resistances of 1-2 MOhm. Unless otherwisespecified, oocytes are voltage clamped at a holding potential of −80 mV.During recordings, oocytes are bathed in continuously flowing (1-3mL/min) medium containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mMMgCl₂, and 5 mM HEPES, pH 7.5 (ND96). Drugs are applied either by localperfusion from a 10 μL glass capillary tube fixed at a distance of 0.5mm from the oocyte, or by switching from a series of gravity fedperfusion lines.

Other oocytes may be injected with a mixture of receptor mRNAs andsynthetic mRNA encoding the genes for G-protein-activated inwardrectifier channels (GIRK1 and GIRK4, U.S. Pat. Nos. 5,734,021 and5,728,535 or GIRK1 and GIRK2) or any other appropriate combinations(see, e.g., Inanobe, A. et. al. (1999) J. of Neurosci. 19(3):1006-1017). Genes encoding G-protein inwardly rectifying K⁺ (GIRK)channels 1, 2 and 4 (GIRK1, GIRK2, and GIRK4) may be obtained by PCRusing the published sequences (Kubo, Y. et al., Nature 364:802-806(1993); Dascal et al., Proc. Natl. Acad. Sci. USA 90:10235-10239 (1993);Krapivinsky et. al., Nature 374:135-141 (1995) and J. Biol. Chem.270:28777-28779 (1995)) to derive appropriate 5′ and 3′ primers. Humanheart or brain cDNA may be used as template together with appropriateprimers.

Heterologous expression of GPCRs in Xenopus oocytes has been widely usedto determine the identity of signaling pathways activated by agoniststimulation (Gundersen, C. B. et al. (1983), Proc. R. Soc. Lond. B.Biol. Sci. 219(1214):103-109; Takahashi et al. (1987), Proc. Natl. Acad.Sci. USA 84(14):5063-5067). Activation of the phospholipase C(PLC)pathway is assayed by applying a test compound in ND96 solution tooocytes previously injected with mRNA for the OSGPR114 or OSGPR78receptor and observing inward currents at a holding potential ofapproximately −80 mV. The appearance of currents that reverse at −25 mVand display other properties of the Ca2+-activated Cl-channel isindicative of receptor-activation of PLC and release of IP₃ andintracellular Ca²⁺. Such activity is exhibited by GPCRs that couple toGq or G₁₁.

Involvement of the Gi/o class of G-proteins in GPCR-stimulatedCa2+-activated Cl-currents is evaluated using PTX, a toxin whichinactivates Gi/o G-proteins. Oocytes are injected with 25 ng PTX/oocyteand modulation of Ca2+-activated Cl-currents by OSGPR114 or OSGPR78receptor is evaluated 2-5 h subsequently.

Elevation of intracellular cAMP can be monitored in oocytes byexpression of the cystic fibrosis transmembrane conductance regulator(CFTR) whose Cl⁻-selective pore opens in response to phosphorylation byprotein kinase A (Riordan, J. R. (1993) Ann. Rev. Physiol. 55: 609-630).In order to prepare RNA transcripts for expression in oocytes, atemplate is created by PCR using 5′ and 3′ primers derived from thepublished sequence of the CFTR gene (Riordan, J. R. (1993) Ann. Rev.Physiol. 55: 609-630). The 5′ primer includes the sequence coding for T7polymerase so that transcripts can be generated directly from the PCRproducts without cloning. Oocytes are injected with 10 ng of CFTR mRNAin addition to 10-15 ng mRNA for OSGPR114 or OSGPR78.Electrophysiological recordings are made in ND96 solution after a 2-3day incubation at 18° C. Currents are recorded under dual electrodevoltage clamp (Axon Instruments Inc.) with 3 M KCl-filled glassmicroelectrodes having resistances of 1-2 Mohm. Unless otherwisespecified, oocytes are voltage clamped at a holding potential of −80 mV.During recordings, oocytes are bathed in continuously flowing (1-3mL/min) medium containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mMMgCl₂, and 5 mM HEPES, pH 7.5 (ND96). Drugs are applied either by localperfusion from a 10 μL glass capillary tube fixed at a distance of 0.5mm from the oocyte, or by switching from a series of gravity fedperfusion lines.

Activation of G-protein G_(i) and G_(o) can be monitored by measuringthe activity of inwardly rectifying K⁺ (potassium) channels (GIRKs).Activity may be monitored in oocytes that have been co-injected withmRNAs encoding the mammalian receptor plus GIRK subunits. GIRK geneproducts co-assemble to form a G-protein activated potassium channelknown to be activated (i.e., stimulated) by a number of GPCRs thatcouple to G_(i) or G_(o) (Kubo, Y. et al. (1993), Nature 364:802-806;Dascal et al. (1993), Proc. Natl. Acad. Sci. USA 90:10235-10239).Oocytes expressing the mammalian receptor plus the GIRK subunits aretested for test compound responsivity by measuring K⁺ currents inelevated K⁺ solution containing 49 mM K⁺.

Results and Discussion

Cloning of the Full Length Sequences of OSGPR114 and OSGPR78

The human OSGPR114 receptor shares the highest amino acid homology withhuman OSGPR78 (36%), and 29% with the P2Y5-like (P2Y9) receptordescribed by Janssens (Janssens et. al. (1997) Biochem. Biophys. Res.Commun. 236(1):106-112). OSGPR78 shared a 56% homology with this P2Y9receptor. OSGPR114 and OSGPR78 receptors are only distantly related toother known LPA receptors. Homology clustering analysis with OSGPR114and OSGPR78 indicates that they align most closely with each other, andthe closest relatives from the known superfamily of GPCR are the P2Ynucleotide receptors, the cysteinyl leukotriene receptors and theplatelet-activating factor (PAF) receptor.

Expression Profiling Analysis

Gene expression analysis, by quantitative RT-PCR, indicates that thereis a wide expression of OSGPR114 in normal human tissues (FIG. 13). Thehighest expression levels (normalized to TFIIB expression) were observedin the kidney, leukocytes, lung and aorta, with lower additionalexpression in the liver, pancreas, adipose tissue, cerebellum,hippocampus, small intestine, trachea, thymus, bladder and ovary.Expression of the receptor in other tissues was low relative to theselevels, but detectable in all tissues tested.

Gene expression analysis, by quantitative RT-PCR indicates that there isa high and almost ubiquitous expression of OSGPR78 in normal humantissues (FIG. 13). High expression levels (normalized to TFIIBexpression) were observed in all tissues tested, and especially high inperipheral tissues and cells involved in the immune system. The prostateshowed the highest expression level in this particular experiment.

It is perhaps not surprising that LPA specific GPCRs are foundextensively throughout the body, due to the known role of LPA inmodulating a vast variety of physiological processes, including growthand differentiation of cells. The distribution patterns of both OSGPR78and OSGPR114 are therefore in correlation with the known biologicalactivities of the activating molecule. Similarly, the observation thatLPA synthesizing enzymes, and LPA binding proteins, are found free inthe circulation and therefore can theoretically deliver LPA to any partof the body, is further consistent with this hypothesis.

A number of human cancer cell lines were also tested for expression ofOSGPR114 and OSGPR78, and found to express the receptors (FIG. 3).Cancer cell lines derived from the following tissues of origin werefound to express OSGPR114: colon (T84, SW480, LoVo and HCT8), breast(DU4775 and T47D), testicular (577MF) and pancreatic (ASPC-1, Cappan2,CFPAC, HPAFII and Panc1). Cancer cell lines derived from the followingtissues of origin were found to express OSGPR78: Brain (T98G), Leukemia(HL60), breast (MDA-MB-435, mcf7, T47D), bladder (AII63, T24), colon(HT29), GI tract (cappan1), pancreatic (ASPC-1, BXPC, Cappan2) and liver(HepG2).

In order to further characterize the expression of OSGPR78 and OSGPR114in tumors, the expression of these receptors was measured in matchedpairs of tumor samples using RT-PCR (FIGS. 4 and 5). The matched pairswere all generated from internal tissue samples, with the exception of acolon matched pair tumor sample purchased directly from Clontech (ID#).Expression of OSGPR78 was observed to be upregulated in 5/8 colonsamples with no change in 3/8. OSGPR78 was also upregulated in 2/7 and2/9 breast and lung samples respectively, with no change observed in theremaining samples. Gene expression of OSGPR114 also showed upregulationin certain matched pairs of tumor samples. OSGPR114 was upregulated in5/9 lung, 4/7 breast and 4/8 colon samples, with no change in the othersamples tested. The demonstration that in over 50% of the cancer matchedpairs tested in this study, OSGPR114 gene expression demonstrates thelikely importance of this receptor to a variety of cancer types. OSGPR78gene expression was also increased in greater than 50% of colon cancerstested, with a smaller percentage of breast and colon tumor samples alsoshowing increased gene expression. These particular cancer types weretested due to the number of studies implicating LPA signaling in colon,lung and breast cancers.

In summary, the pattern of gene expression of OSGPR114 and OSGPR78 inmalignant cell lines, and in particular, the over-expression of thereceptor gene in matched pairs of tumor sample RNAs strongly implicatesthese genes in the disease of cancer. This observation is consistentwith previous demonstrations of increased LPA signaling in cancer. It istherefore highly plausible that the increased LPA signaling seen incarcinogenesis and disease progression is the result of increased LPAsignaling through OSGPR114 and OSGPR78 and that moieties which can blockthe effects of LPA at these receptors would be of therapeutic benefitfor the treatment of a range of cancer types. Such receptor antagonistsmay take the form of small molecules, peptides or antibodies that blockthe receptor directly, or a downstream target in the OSGPR114 and/orOSGPR78 signaling pathway.

Yeast-Based Reporter Assays

Co-expression of the receptor and the reporter genes in yeast imparts atriple selection benefit to the yeast, allowing them to grow in aLEU-URA-TRP deficient media. Colonies that grew on SD-LUT(−) plates, andtherefore expressed the plasmids of interest, were tested in thefluorogenic β-galactosidase assay. Due to the homology clusteringanalysis that indicated that the closest relatives of this orphan GPCRwere nucleotide receptors, PAF receptor and leukotriene receptors,various nucleotides, leukotrienes, PAF and related endogenously foundbiologically active lipids were tested at OSGPRs 114 and 78.Additionally, the prior art had suggested that the nucleotide ADPspecifically activates this receptor, and so this compound was alsotested in the assay. ADP and ATP were found to have no effect in theyeast-based assay, nor did other nucleotides, PAF, leukotrienes andvarious other phospholipids and lysophospholipids. However,lysophosphatidic acid (LPA), with a number of lipid backbones(myristoyl, palmitoyl, stearoyl and oleoyl) were all shown to cause anincrease in fluorescence in yeast transformed with OSGPR114 or OSGPR78receptor (FIGS. 6 and 7), but not in vector transformed cells. OleoylLPA was the most efficacious and potent of the compounds tested in boththe OSGPR78 assay and the OSGPR114 assay.

Multiple yeast strains with different G-protein chimeras were alsotested for optimal response to oleoyl LPA and several but not allstrains responded to ligand induction. This is normal. Generally, the Galpha chimeras have been designed to mimic mammalian receptors asclosely as possible. In a mammalian system, receptors which couple toGalpha i/o family generally will not respond in a Galpha q basedread-out. It was found that using the yeast assay chimeras of Galpha q(20156), and Galphas (20202) could produce a response to oleoyl LPA inOSGPR114-transformed yeast. Members of the Galpha i and Galpha 12/13family chimeras were optimal for LPA response in OSGPR78-transformedyeast (13393, 15074, 13395) (FIG. 6). 20156 and 20202 represent internalnomenclature for yeast expressing sandwich chimeras of the G alpha q ands families and 13393 represents a Galpha chimera which contains the GPA1with the five terminal amino acids of mammalian Galpha i2. 13395represents GPA1 with the five terminal amino acids from mammalianGalpha12 and 15074 represents GPA1 with the five terminal amino acidsfrom mammalian Galpha13. The sandwich chimers represent N- andC-terminal sections of the mammalian Galpha, fused to a middle sectionof the endogenous Galpha from yeast (GPA1). Vector controls in the sameyeast strains did not respond to oleoyl LPA, nor did other GPCRexpressed in the comparable chimeric yeast backgrounds. Comparisonbetween the OSGPR78 and OSGPR114 demonstrate that, despite therelatively low homology between the receptors, the pharmacology of thereceptors was almost identical with respect to the compounds tested inthis study.

In summary, OSGPR114 and OSGPR78, when expressed in yeast respond toLPA. This is in contrast to some previous studies (Webb et. al. (1996)Biochem. Biophys. Res. Commun. 219(1):105-110) that suggested thepossibility that certain nucleotides may activate OSGPR78. Thispossibility was not confirmed in our studies, or in other publishedstudies (Li et. al. (1997) Biochem. Biophys. Res. Commun.236(2):455-60). In terms of efficacy and potency, the optimalendogenously produced agonist tested oleoyl-LPA. The identification ofnovel receptors that are activated by oleoyl LPA, and other LPA analogs,allows for a speculation as to the potential role of these receptors inthe body. Based on our understanding of the physiological effects of LPAand its role in cell growth, differentiation, motility, metastasis,invasion, artherosclerosis, obesity, cardiovascular and respiratoryregulation, OSGPR114 and OSGPR78 represent molecular mechanisms by whichthese effects of LPA in the body may be mediated and therefore act asunique targets for therapeutic intervention in the relevant diseases.

Mammalian Cell Based Assays

Radioligand Binding

Stable transfection of CHO cells with OSGPR114 led to increased specific[3H] oleoyl LPA binding (14 nM) compared to CHO cells transfected withvector alone. The CHO cell membranes control did however demonstratespecific LPA binding. However, when the data are normalized to the CHOcells, the CHO-OSGPR114 membranes demonstrated a significant level ofspecific LPA binding, suggesting that the radiolabeled LPA was bindingto OSGPR114. The CHO-OSGPR114 clone which exhibited the highest level ofLPA specific binding, which was also the clone that exhibited thehighest level of OSGPR114 mRNA expression was used for subsequentexperimentation. The demonstration that OSGPR114 stably transfect CHOcells exhibit significantly higher specific [3H] LPA binding extends theobservation from the yeast-based assays that LPA binds to OSGPR114. Thedata also demonstrate that CHO cells contain an as yet unidentified formof LPA receptor, which may or may not be one of the known LPA receptors(the hamster homolog).

B) GTPγS Binding

Membranes prepared from the CHO and CHO-OSGPR114 stable transfectantswere tested for responses to LPA in the GTPγS binding assay. The GTPγSbinding assay is an assay that has proved of high utility for measuringthe activation, and antagonism, of GPCR, particularly those receptorswhich couple to the Gi/o family of G-proteins. Oleoyl LPA, atconcentrations from 1 nM to 300 nM was applied to membranes from each ofthe cell lines. LPA produced concentration-response curves in both CHOmembranes and in the stably transfected CHO-OSGPR114 membranes. Theobservation that the CHO parental cell also produces a LPA dependentconcentration response curve in the GTPγS binding assay confirms thatthe unidentified receptor shown to be present in these membranes in theradioligand binding assay is a GPCR. However, the EC50 value of LPAcalculated in the CHO-OSGPR114 stable transfectants was significantlydifferent to the parental CHO membranes, suggesting that LPA activatedOSGPR114, as well as the endogenous CHO receptor, and with a higherpotency. The EC50 values in the CHO-OSGPR114 and CHO membranes were 137and 768 nM respectively, with comparable Emax values.

C) FLIPR Assay

CHO, or CHO-OSGPR114, cells were tested in a FLIPR assay, a techniquefrequently used to measure changes in intracellular calciumconcentrations in response to GPCR activation. LPA was added to thecells in the presence or absence of pertussis toxin. Pertussis toxin isan agent known to ADP-ribosylate and thus inactivate members if the Gi/ofamily of G-proteins and has been frequently used to assess whetherG-proteins of this family are involved in the signal transductionpathway of a particular GPCR. In the FLIPR assay, no significant changein intracellular calcium concentration was observed on response to LPAadministration in the CHO cells. However, LPA caused aconcentration-dependent response in the CHO-OSGPR114 cells with an EC50of 59 nM, a figure in close agreement with that observed in the GTPγSbinding assay. Co-addition of pertussis toxin completely abolished thiseffect. This result demonstrates that activation of OSGPR114 by LPA cancause an increase in intracellular calcium concentration, an effectmediated by G-proteins of the Gi/o family as indicated by thesensitivity to pertussis toxin.

In summary, the results of the cell and membrane-based assays usingexpression of OSGPR114 in mammalian cells further confirms thespecificity of the response observed in yeast, that OSGPR114 is a novelspecific LPA receptor. Interestingly, most of the mitogenic andmotogenic responses of LPA in the majority of cell-based assays havedemonstrated that the effects of LPA on proliferation and motility areregulated by Gi/o coupled receptors. In proving that OSGPR114 also is aGi/o coupled LPA-specific receptor lends further weight to the proposalthat OSGPR114 is likely to be an important molecular regulator of LPAeffects on cell proliferation and motility, enhancing the rationale forits use as a target for anti-cancer therapeutics.

OSGPR114 and OSGPR78 could not have been predicted to be activated byLPA through analysis of the DNA or amino acid sequences of thereceptors. They have a very low homology to the known LPA receptors(LPA1-3) and homology-clustering analysis of known and orphan GPCRs, atechnique that can be predictive of ligand-receptor coupling systems(Joost and Methner, (2002) Genome Biol. 3(11): research 0063.1-0063.16(at GenomeBiology website); Vaidehi et. al. (2002) Proc. Natl. Acad.Sci. USA. 99(20):12622-12627), does not demonstrate any predictablerelationship between the known LPA receptors and OSGPR114 and OSGPR78.Indeed, a clustering analysis demonstrates that the two closely relatedGPCR OSGPR114 and OSGPR78 receptors cluster with nucleotide receptorssuch as the P2Y family and the PAF receptor.

Role of OSGPR114 and OSGPR78 in Cancer Cells

Assessment of the validity of OSGPRs 114 and 78 as cancer targets wasapproached in three ways. Firstly, in cell lines shown to express thereceptors, the effects of LPA on growth and survival was assessed.Secondly, small interfering RNAs (siRNA) specific for the receptors wasintroduced to cells expressing OSGPR114 and/or OSGPR78 and the effect oncell proliferation/survival measured. Finally, in cells known to expressthe receptors, the signaling pathways activated by LPA wereinvestigated.

Summary of Results:

In serum-free conditions, LPA induced cellular proliferation andprevented apoptosis of H CT-8 cells, a colon cancer cell line known toexpress high levels of OSGPR114.

i) Specific siRNAs targeting OSGPR114 inhibited cellular proliferationof all cell lines tested. This growth inhibition was dependent only onthe expression of OSGPR114 and not on the presence of other known LPAreceptors in these cells. Associated with this growth inhibition in mostof the cell lines was an induction of apoptosis.

ii) Specific siRNAs targeting OSGPR78 inhibited cellular proliferationin most cell lines tested. Within a subset of these cell lines,apoptosis was also induced by the introduction of OSGPR78 siRNA

3) In HCT-8 cells, and using activation-specific antibodies, LPA wasshown to activate multiple growth and survival pathways, includingERK1/2, Akt, paxillin, Shc, and SHP-2. Transactivation of thereceptor-tyrosine kinase, EGFR, was also observed.

Part I: Growth/Survival Effects of LPA in HCT-8 Cells:

The ability of LPA to act as a growth factor, and a survival factor forcells in culture, is well characterized in the literature and is thesubject of numerous reviews (For review See Mills and Moolenaar (2003)Nat. Rev. Cancer 3:582-91). Due to the acknowledged presence of threeother specific LPA receptors (LPA1-3) and the demonstrated ability ofthese receptors to transduce some of these growth/survival effects ofLPA on cells, it was of interest to determine OSGPR114 may also possesssimilar biological effects. HCT-8 cells were chosen for this experimentas they have one of the highest expression levels of OSGPR114 within thecell lines expression profiled. Additionally, OSGPR114 is thepredominant receptor, although Edg4 is also expressed in these cells atmuch lower levels

The effect of LPA on cell proliferation (Cell TiterGlo assay, Promega)and induction of apoptosis (Apo-One Caspase 3/7 assay, Promega) wasmeasured in the presence and absence of serum. Cell-titer Glo is anassay that quantitates ATP as a measurement of the number of viablecells in a culture. The assay utilizes a luciferase-based luminescentsignal. The Apo-One assay utilizes a profluorescent caspase 3/7consensus substrate to measure the activation of caspase in the sample.The experiments were conducted in serum-free media as it is known thatserum contains high concentrations of LPA (of the order of 10-30 uM).Briefly, cells were plated in 96-well plates at a density of 20,000cells/well. After 24 h the serum-containing media was removed from thecells and replaced with either serum-containing or serum-free media.After 24 h LPA, at concentrations from 1-100 μM was added to the cells.24 h later the induction of apoptosis and the level of cellularproliferation was measured using the Cell titer-glo or Caspase 3/7Apo-One assay kits commercially available from Promega. The data areshown in FIG. 1.

LPA acting on HCT-8 cells induced growth of the cells in a concentrationdependent manner in serum free conditions, and reversed apoptosisinduced through the serum starving of the cells. Due to the highconcentration of LPA in serum, no statistically significant effects ofLPA were observed in the presence of 10% FCS.

Part II: Effects of OSGPR114 and OSGPR78 siRNAs on Cell Growth andSurvival:

The sequence-specific gene silencing induced by double stranded RNA isknown as RNA interference (RNAi). It is a powerful technique with whichthe function of a specific gene can be investigated through itssilencing by small interfering RNAs (siRNA). Because of the sequencespecificity of the siRNA, only the gene of interest should beeffectively silenced. It is a particularly powerful technique forassessing knockdown/inhibition of a gene's function in the absence of apharmacological inhibitor of the gene (For review see Devereaux et al.(2003) Seminars Cancer Biol. 13:293-300). In order to assess the role ofthe novel LPA receptors OSGPR114 and OSGPR78, siRNA oligonucleotides(SmartPool) specific to either OSGPR114 or OSGPR78 were provided byDharmacon Inc. Non-specific control oligonucleotides were used asnegative controls and siRNA targeting polo-like kinase 1 (PLK1) was usedas a positive control as siRNA targeting of this gene has been shown inthe literature to induce apoptosis and inhibit cell growth.

With conditions and reagents that were optimized for each cell linetested, the siRNAs (50-75 nM) were introduced into cells known toexpress a particular receptor of interest. Such optimization includedvarying the Transfection reagent, the oligonucleotide concentration, thelipid:oligonucleotide ratio, the optimal transfection cell density andthe time for optimal gene knockdown. Every cell line displayed its ownunique optimal transfection condition profile.

The effects of this transfection upon cell proliferation were measuredusing a fluorometric 5-bromo-2-deoxyuridine (BrDU) assay (Roche) or aCell-titer Glo assay (Promega) and the effects upon apoptosis bymeasuring caspase 3/7 cleavage using the Apo-One caspase kit (Promega).Manufacturers instructions were followed with each kit and either 48 hor 72 h post-transfection endpoints were tested.

The cell lines chosen were picked upon the basis of a demonstration ofthe expression of the receptor of interest as measured by quantitativeRT-PCR. RT-PCR was conducted as previously described. Table 1 shows therelative expression of OSGPR78 or OSGPR114 in cell lines used for thesiRNA experiments.

OSGPR114 Cell Line Origin Expression OSGPR78 expression HCT-8 ColonCancer 0.15* 0.0016* KLE Endometrial cancer 1.48E−03 4.80E−04 HCT-116Colon cancer 3.60E−04 2.30E−05 A2058 Melanoma 0*   0.641*  H460 Lungcancer 1.90E−07 3.50E−05 MDAH-2774 Ovarian cancer 4.30E−04 1.07E−03 Dataexpressed relative to GAPDH expression except where denoted by * (Dataexpressed relative to TFIIB)

Table 1. Expression of OSGPR78 and OSGPR114 in cells used for siRNAexperiments (N.B. E denotes exponent often; e.g. 1.48E-03=1.48×10⁻³)

FIGS. 2-7 show a summary of the siRNA experiments conducted withOSGPR114 and OSGPR78 specific siRNAs, and the effects of thetransfection of these oligonucleotides on cell proliferation andsurvival in cell lines of interest. All data are expressed as a fractionof (for BrDU/Cell titer glo assays) or fold induction over (for caspaseassays) untreated cells. Negative controls (non-specific siRNA) andpositive controls (polo-like kinase 1 (PLK1) siRNA) are also shown. PLK1was used as a positive control as PLK1 siRNA has been demonstrated inthe literature to induce apoptosis and inhibit cell growth(Spankuch-Schmitt et al. (2002) J. Nat. Cancer Inst. 94:1863-1877). Thetransfection reagent, concentration of siRNA oligonucleotide, originalcell plating density and the n number are shown in the headers for eachgraph. A single n number was a single experiment conducted in triplicateand the data averaged. The timepoint used for data generation (either 48or 72 h post-siRNA transfection is also shown in individual graphtitles. Statistical significance is defined as *p<0.05 and ***p<0.001(One way ANOVA, Dunnett's post-hoc).

Table 2 demonstrates a summary of the effects of OSGPR114 and OSGPR78siRNAs on growth and proliferation in each of the cell lines tested inthis study. An indication of yes illustrates a statistically significantinhibition of cell proliferation or a statistically significantinduction of apoptosis.

OSGPR114 OSGPR78 Cancer Cell Line Proliferation Apoptosis ProliferationApoptosis HCT-8 (colon) Yes Yes Not tested KLE Yes No No No(endometrial) A2058 Not tested Yes Yes (melanoma) HCT116 (colon) Yes YesNo No H460 (lung) Yes Yes Yes Yes MDAH-2774 Yes No Yes No (ovarian) (n =1)

Table 2: Comparison of data from FIGS. 2-7 showing a summary of theeffects on proliferation and apoptosis of OSGPR114 and OSGPR78 siRNAtransfection in 6 cell lines.

Summary of siRNA Experiments:

In summary, introduction of siRNAs specific to OSGPR114 induced growthinhibition in five out of five cell lines tested, and induced apoptosisin three of the five cell lines strongly suggesting that this receptoris involved in promoting growth and survival of all cancer cell linestested. Considering that every cancer cell line tested, derived from amultitude of original tumors, were sensitive to the growth inhibitoryeffects of OSGPR114 knockdown, it is likely that additional cell lines,from multiple other cancer types will respond similarly. Additionally,the introduction of siRNAs specific to OSGPR78 also inhibited the growthand induced apoptosis in the cell lines in which it was tested, alsoimplicating this receptor as a mediator of growth and survival ofcertain cancer cells in culture. Again, and similar to the previouslystated hypothesis, it is likely that the proliferation of multiple othercancer cell lines, and tumor types, will be sensitive to geneticknockdown of OSGPR78, and that the sensitivity will only be dictated bythe expression of the receptor in a particular cancer cell. Furthermore,these data demonstrate that pharmacological inhibitors of the receptors(acting by causing, for example, reduced receptor expression level,reduced receptor activity level, or decreased receptor-stimulated signaltransduction activity) will also cause growth inhibition and induceapoptosis in cells that express these receptors. This would allow theuse of pharmacological inhibitors of these receptors in disease stateswhere an inhibition of cellular growth and/or an induction of cellularapoptosis would be beneficial, for example for the treatment of cancers.

Part III: LPA Signaling Pathways in HCT-8 Cells:

Introduction:

Many of the genes and signaling pathways that are implicated inoncogenic transformation and tumor growth and survival are known. Thesehave been increasingly well characterized in the public literature overthe last 2-3 decades. It was therefore of interest to determine whatrole OSGPR114 may play in regulating these signaling pathways. In orderto determine the roles of the receptor, the effect of LPA on thesepathways in cells known to express OSGPR114 was tested. In defining thecancer cell line to use for these studies multiple cancer cell lineswere expression profiled not only for OSGPR114 expression but also theexpression of other LPA receptors. LPA is known to be a critical growthand survival factor for virtually every cancer cell in culture not leastbecause almost all cells express a multiplicity of LPA receptors. Forexperiments investigating the signaling pathways of OSGPR114 it wastherefore important to use a cancer cell which expressed OSGPR114 at thehighest relative levels to other LPA receptors. HCT-8 colon cancer cellswere judged to be the best model available for these experiments and agraphical representation of the expression profile of all LPA receptorsin these cells is shown in FIG. 9. They were judged to be the best modelbased on the very high level of OSGPR114 mRNA expressed by these cells,in addition to the lower relative levels of other known LPA receptors inthese cells.

ERK Activation:

One of the principal proliferative signaling pathways is the activationof ERK. Multiple growth factor receptors have been shown to inducecellular proliferation as a result of activation of this pathway. LPAhas been demonstrated in many cell lines, and acting at different LPAreceptors, to activate ERK. However, the ability of LPA acting atOSGPR114 to effect ERK is unknown. HCT-8 cells, which express OSGPR114at high levels, provide one of the best models available. LPA was foundto stimulate ERK in HCT-8 (and ASPC-1, another cell line shown toexpress OSGPR114) cells. This evidence supporting the hypothesis thatLPA, likely acting at least in part at OSGPR114, induces ERK activation,a signaling pathway that is known to stimulate cellular proliferation.

EGFR Transactivation:

The ability of LPA to phosphorylate ERK1/2 has been extensivelydescribed in the literature, and three principal mechanisms have beendescribed for this effect. Firstly, LPA receptors have been shown tostimulate ERK via a Gα12/13-Rho based pathway. Additionally, thisactivation of ERK has been shown to be dependent on two differentreceptor tyrosine kinase (RTK) transactivation pathways. The first ofthese pathways involves β-arrestin recruitment and activation of Src,which then transactivates the RTK. The second of these pathways involvesShc which is recruited by LPA and can itself activate membrane-boundmetalloproteases (e.g. ADAM10) which causes release of heparin-boundepidermal growth factor (EGF) which can then activate EGF receptor. EGFand activation of the EGF receptor is a well known proliferative pathwayfor many cell lines. The possibility of LPA causing transactivation ofEGFR in HCT-8 cells was tested as the EGFR signaling pathway is known tobe present and active in these cells. From FIG. 11, it is clear thatLPA, at a concentration of 10 μM is capable of transactivating EGFR inHCT-8 cells. From the LPA-based literature, the potential for LPA toalso transactivate other RTKs, such as PDGFR for example, in these cellsis a very likely possibility.

Additional Signaling Pathways Utilized by LPA in HCT-8 Cells:

Many of the growth and survival signaling pathways of cancer cells havebeen identified. In addition to the stimulation of the mitogen-activatedprotein kinase pathway demonstrated by the activation of ERK1/2 by LPA,and the transactivation of other known cancer cell growth factorreceptors such as EGFR, the effect of LPA on additional known growth andsurvival signaling was investigated. The protein Shc is an adaptorprotein and the protein phosphatase SHP-2 are known to link theactivation of multiple growth factor receptors to their downstreamsignaling pathways in cancer cells (For review see Yart et al. (2003)Current Cancer Drug Targets 3:177-192). These proteins are involved inmitogenic signaling by linking growth stimuli to theRas-mitogen-activated protein kinase cascade and the PI3K survivalpathway. LPA, at a concentration of 10 uM when added to HCT-8 cells,induced the phosphorylation of Shc and SHP-2, further demonstrating therole of LPA receptors in HCT-8 cells acting as growth and survivalfactors. Additionally, paxillin is a scaffold molecule intricatelyinvolved in the regulation of focal adhesions in cancer cells. It istherefore involved in the regulation of cell spreading and motility andis controlled by a multitude of signaling moieties including growthfactors, and their downstream signaling molecules. Paxillin recruitsthese signaling components into specific cell compartments known asfocal adhesions (For review, see Schaller (2001) Oncogene 20:6459-72).LPA, acting at other LPA receptors, has been shown in the literature toincrease cellular motility as well as increasing cellular growth andsurvival. By treating HCT-8 cells with LPA (10 uM), the effect of LPA onthe phosphorylation of paxillin was measured using activation statespecific antibodies (FIG. 12). It was found that LPA caused a highlysignificant increase in phosphorylated paxillin, further supporting itsrole not only as a growth and survival factor but also as a regulator ofcancer cell motility.

LPA and Akt:

The protein Akt is a well characterized survival pathway for many cancercells and is a pathway that can be induced by multiple cell growthfactors (For review, see Marte and Downward (1997) Trends Biochem. Sci.22:355-358). FIG. 13 demonstrates the ability of LPA, at a concentrationof 10 uM to stimulate phosphorylation of Akt, as detected by aphospho-specific Akt antibody. The cells used for this experiment were aCHO cell line stably transfected with OSGPR114. The cells were treatedwith vehicle, LPA (10 uM) or EGF (1 uM) for 3 minutes. This experimentdemonstrates the ability of LPA, in cells stably over-expressingOSGPR114, to activate this well known survival pathway in cells.

SUMMARY

LPA has been shown to cause the proliferation of HCT-8 cells inserum-free conditions and to prevent apoptosis induced by serumstarving. Additionally, siRNA has shown that biochemical inhibition ofOSGPR114 and OSGPR78 both inhibits cancer cell proliferation and inducesapoptosis. Finally LPA has been shown to stimulate multiple signalingpathways implicated in the growth, survival and motility of cancer cellsincluding ERK1/2, EGFR, Akt, Shc, SHP-2 and paxillin. The activity ofLPA in HCT-8 cells, a colon cancer cell line known to highly expressOSGPR114, in inducing all of these pathways lends further support to thehypothesis demonstrated with the siRNA and growth measurementexperiments that LPA is an important growth and survival factor forcancer cells in vitro, and demonstrates that a pharmacological inhibitorof LPA signaling at OSGPR114 will be beneficial in the treatment ofcancer and other proliferative diseases.

INCORPORATION BY REFERENCE

All patents, published patent applications and other referencesdisclosed herein are hereby expressly incorporated herein by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1. An assay process for identifying a compound that specifically bindsto an OSGPR114 receptor, wherein said OSGPR114 receptor comprises anamino acid sequence having at least 95% identity to SEQ ID NO:3, thatcan induce oleoyl lysophosphatidic acid stimulation of G-proteinactivity in cells, said process comprising: providing either (a) twosamples of cells expressing on their cell surface the OSGPR114 receptor,or (b) two samples of a membrane preparation from said cells; contactingone sample with a lysophosphatidic acid ligand known to bind to thereceptor, under conditions suitable for binding of said ligand to thereceptor, in the presence of a test compound; contacting the secondsample with said ligand, under conditions suitable for binding of saidligand to the receptor, in the absence of the test compound; measuringthe specific binding of the ligand to the receptor in the presence ofthe compound; measuring the specific binding of the ligand to thereceptor in the absence of the compound; and comparing the binding inthe presence and in the absence of the compound being tested, wherein adifference in comparison indicates that the compound binds to theOSGPR114 receptor.
 2. The process of claim 1, wherein thelysophosphatidic acid has a fatty acid group with a carbon chain lengthselected from C14, C15, C16, C17, C18, C19, C20, C21 and C22.
 3. Theprocess of claim 1, wherein the lysophosphatidic acid is selected frommyristoyl lysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid.
 4. Theprocess of claim 1, wherein the lysophosphatidic acid is selected from1-myristoyl lysophosphatidic acid, 1-oleoyl lysophosphatidic acid,1-palmitoyl lysophosphatidic acid, and 1-stearoyl lysophosphatidic acid.5. The process of claim 1, wherein the lysophosphatidic acid has analkyl ether or alkenyl ether group.
 6. The process of claim 5, whereinthe alkyl ether or alkenyl ether group has a carbon chain lengthselected from C14, C15, C16, C17, C18, C19, C20, C21 and C22.
 7. Theprocess of claim 1, wherein the cells are insect cells, mammalian cells,human cells, or yeast cells.
 8. The process of claim 1, wherein thecells are NIH-3T3, mouse Y1, CHO, RH7777, Jurkat, HCT4, RBL243, HeLa,ASPC-1, HEK-293, or COS7 cells.
 9. The process of claim 1, wherein theOSGPR114 receptor comprises an amino acid sequence having SEQ ID NO:3,that can induce oleoyl lysophosphatidic acid stimulation of G-proteinactivity in cells.
 10. A method of screening a plurality of compoundsnot known to bind to an OSGPR114 receptor, wherein said OSGPR114receptor comprises an amino acid sequence having at least 95% identityto SEQ ID NO:3, that can induce oleoyl lysophosphatidic acid stimulationof G-protein activity in cells, to identify a compound whichspecifically binds to the OSGPR114 receptor, said process comprising:providing either (a) two samples of cells expressing on their cellsurface the OSGPR114 receptor, or (b) two samples of a membranepreparation from said cells; contacting one sample with alysophosphatidic acid ligand known to bind to the receptor, underconditions suitable for binding of said ligand to the receptor, in thepresence of the plurality of compounds not known to bind to thereceptor; contacting the second sample with said ligand, underconditions suitable for binding of said ligand to the receptor, in theabsence of the plurality of compounds; measuring specific binding of theligand to the receptor in the presence of the plurality of compounds;measuring specific binding of the ligand to the receptor in the absenceof the plurality of compounds; comparing the binding in the presence andin the absence of the plurality of compounds, wherein a difference inthe compared binding results indicates that one or more compounds in theplurality of compounds binds to the OSGPR114 receptor; and determining,when a difference in the compared binding is found, the binding to theOSGPR114 receptor of each compound included in the plurality ofcompounds, to identify any compound included therein which specificallybinds to the OSGPR114 receptor.
 11. The method of claim 10, wherein thelysophosphatidic acid has a fatty acid group with a carbon chain lengthselected from C14, C15, C16, C17, C18, C19, C20, C21 and C22.
 12. Themethod of claim 10, wherein the lysophosphatidic acid is selected frommyristoyl lysophosphatidic acid, oleoyl lysophosphatidic acid, palmitoyllysophosphatidic acid, and stearoyl lysophosphatidic acid.
 13. Themethod of claim 10, wherein the lysophosphatidic acid is selected from1-myristoyl lysophosphatidic acid, 1-oleoyl lysophosphatidic acid,1-palmitoyl lysophosphatidic acid, and 1-stearoyl lysophosphatidic acid.14. The method of claim 10, wherein the lysophosphatidic acid has analkyl ether or alkenyl ether group.
 15. The method of claim 14, whereinthe alkyl ether or alkenyl ether group has a carbon chain lengthselected from C14, C15, C16, C17, C18, C19, C20, C21 and C22.
 16. Amethod of claim 10, wherein the cells are mammalian cells, human cells,or yeast cells.
 17. The method of claim 10, wherein the cells areNIH-3T3, mouse Y1, CHO, RH7777, Jurkat, HCT4, RBL243, HeLa, ASPC-1,HEK-293, or COS7 cells.
 18. The method of claim 10, wherein the OSGPR114receptor comprises an amino acid sequence having SEQ ID NO:3, that caninduce oleoyl lysophosphatidic acid stimulation of G-protein activity incells.
 19. A process for determining whether a chemical compoundspecifically binds to and modulates activation of an OSGPR114 receptor,wherein said OSGPR114 receptor comprises an amino acid sequence havingat least 95% identity to SEQ ID NO:3, that can induce oleoyllysophosphatidic acid stimulation of G-protein activity in cells, saidprocess comprising: providing two samples of cells expressing on theircell surface the OSGPR114 receptor, wherein activation of the receptorproduces a second messenger response; contacting one sample, in thepresence of a test compound, with a second compound known to activatethe receptor, under conditions suitable for activation of the receptor;contacting the second sample, in the absence of the test compound, withthe second compound known to activate the receptor, under conditionssuitable for activation of the receptor; measuring the second messengerresponse in the presence of the test compound, measuring the secondmessenger response in the absence of the test compound; and comparingthe second messenger response in the presence and in the absence of thecompound being tested, wherein a difference in the second messengerresponse from the OSGPR114 receptor indicates that the compoundmodulates activation of a OSGPR114 receptor.
 20. The process of claim19, wherein the second compound is a lysophosphatidic acid.
 21. Theprocess of claim 20, wherein the lysophosphatidic acid has a fatty acidgroup with a carbon chain length selected from C14, C15, C16, C17, C18,C19, C20, C21 and C22.
 22. The process of claim 20, wherein thelysophosphatidic acid is selected from myristoyl lysophosphatidic acid,oleoyl lysophosphatidic acid, palmitoyl lysophosphatidic acid, andstearoyl lysophosphatidic acid.
 23. The process of claim 20, wherein thelysophosphatidic acid is selected from 1-myristoyl lysophosphatidicacid, 1-oleoyl lysophosphatidic acid, 1-palmitoyl lysophosphatidic acid,and 1-stearoyl lysophosphatidic acid.
 24. The process of claim 20,wherein the lysophosphatidic acid has an alkyl ether or alkenyl ethergroup.
 25. The process of claim 24, wherein the alkyl ether or alkenylether group has a carbon chain length selected from C14, C15, C16, C17,C18, C19, C20, C21 and C22.
 26. The process of claim 19, wherein thesecond messenger response comprises chloride channel activation, achange in intracellular calcium ion levels, a release of inositolphosphate, a release of arachidonic acid, GTPγS binding, activation ofMAP kinase, cAMP accumulation, a change in intracellular potassium ionlevels, or a change in intracellular sodium ion levels.
 27. The processof claim 26, wherein the second messenger response is measured by achange in reporter gene activity.
 28. The process of claim 27, whereinthe reporter gene is selected from secreted alkaline phosphatase,luciferase, and β-galactosidase.
 29. The process of claim 19, whereinthe OSGPR114 receptor comprises an amino acid sequence having SEQ IDNO:3, that can induce oleoyl lysophosphatidic acid stimulation ofG-protein activity in cells.
 30. A method of preparing a compositioncomprising a compound which specifically binds to an OSGPR114 receptor,wherein said OSGPR114 receptor comprises an amino acid sequence havingat least 95% identity to SEQ ID NO:3, that can induce oleoyllysophosphatidic acid stimulation of G-protein activity in cells, by aprocess comprising: providing either (a) two samples of cells expressingon their cell surface the OSGPR114 receptor, or (b) two samples of amembrane preparation from said cells; contacting one sample with alysophosphatidic acid ligand known to bind to the receptor, underconditions suitable for binding of said ligand to the receptor, in thepresence of a test compound; contacting the second sample with saidligand, under conditions suitable for binding of said ligand to thereceptor, in the absence of the test compound; measuring specificbinding of the ligand to the receptor in the presence of the compound;measuring specific binding of the ligand to the receptor in the absenceof the compound; and comparing the binding in the presence and in theabsence of the compound being tested, wherein a difference in thebinding of the ligand to the OSGPR114 receptor indicates that thecompound binds to the OSGPR114 receptor; and admixing the compound soidentified, or a functional analog or homolog of said compound, with acarrier, thereby preparing said composition.
 31. The method of claim 30,wherein the lysophosphatidic acid has a fatty acid group with a carbonchain length selected from C14, C15, C16, C17, C18, C19, C20, C21 andC22.
 32. The method of claim 30, wherein the lysophosphatidic acid isselected from myristoyl lysophosphatidic acid, oleoyl lysophosphatidicacid, palmitoyl lysophosphatidic acid, and stearoyl lysophosphatidicacid.
 33. The method of claim 30, wherein the lysophosphatidic acid isselected from 1-myristoyl lysophosphatidic acid, 1-oleoyllysophosphatidic acid, 1-palmitoyl lysophosphatidic acid, and 1-stearoyllysophosphatidic acid.
 34. The method of claim 30, wherein thelysophosphatidic acid has an alkyl ether or alkenyl ether group.
 35. Themethod of claim 34 wherein the alkyl ether or alkenyl ether group has acarbon chain length selected from C14, C15, C16, C17, C18, C19, C20, C21and C22.
 36. The method of claim 30, wherein the OSGPR114 receptorcomprises an amino acid sequence having SEQ ID NO:3, that can induceoleoyl lysophosphatidic acid stimulation of G-protein activity in cells.